9.1

• MacConkey-Positive, Oxidase –Negative

  • Enterobacteriaceae
  • Acinetobacter
  • Streptophomonas

  
• True intestinal pathogens (Enterobacteriaceae)

  • Salmonella
  • Shigella
  • Yersinia
  • Escherichia

  
• Opportunistic pathogens (Enterobacteriaceae)

  • Escherichia
  • Klebsiella
  • Enterobacter
  • Citrobacter
  • Proteus
  • Edwardsiella
  • Providencia
  • Morganella
  • Hafnia
  • Serratia

  
• Enterobacteriaceae

  • Gram negative bacilli/coccobacilli
  • Facultative anaerobe
  • Non-spore forming
  • Motile with peritrichous flagella except Tatumella (polar flagella) and Shigella & Klebsiella (nonmotile)
  • Predominantly facultative flora of the human bowel/gastrointestinal tract
  • Some are agents of zoonoses

  
• Enterobacteriaceae (Biochemical Characteristics)

  • Oxidase (-) except Plesiomonas
  • Catalase (+) except Shigella dysenteriae type 1
  • Reduces Nitrates to Nitrite
  • All members ferment glucose

  
• Enterobacteriaceae (Virulence Factors)

  • Ability to adhere, colonize, produce toxins, and invade tissue
  • Some harbor plasmids that can provide antimicrobial resistance genes
  • Possess O (somatic), H (flagellar), and K (capsular) antigens
  • Endotoxins present within the cell walls are responsible for much of the morbidity and mortality resulting from infections

  
• Enterobacteriaceae (Colonies on BAP)


  • Luxurious, moist, smooth, gray, shiny, entire, convex and opaque

  
• Enterobacteriaceae (Categories)

  • Opportunistic pathogens
  • Primary pathogens (true pathogens like Salmonella, Shigella, and Yersinia)

  
• MacConkey agar

  Selective and differential agar that contains crystal violet dye and neutral red pH indicator. Lactose fermentation results in acid production and pink/red colonies.

  
• Oxidase test


  Cytochrome oxidase participates in electron transport and in the nitrate metabolic pathways of certain bacteria. 1% tetramethyl-p-phenylenediamine dihydrochloride reagent is used.

  
• Culture media

  • Eosin Methylene Blue (EMB)
  • MacConkey agar
  • Salmonella Shigella agar (SSA)
  • Hektoen Enteric agar (HEA)
• Triple Sugar Iron (TSI) test

  1. Initial step in Enterobacteriaceae identification

  2. Contains peptone, 3 sugars (lactose, sucrose, glucose), phenol red pH indicator, and ferrous sulfate H2S indicator

  3. Slant and butt reactions indicate sugar fermentation and H2S production

  
• Lysine Iron Agar (LIA) test


  1. Determines if an organism can deaminate lysine (aerobic, slant) and decarboxylate lysine (anaerobic, butt)

  2. Also detects H2S production
• IMViC tests

  • Indole
  • Methyl red
  • Voges-Proskauer
  • Citrate
• The IMViC tests are used to further identify Enterobacteriaceae

  
• H2S

  Slant/A
• BUTT


  Lysine decarboxylaton
• (+)

  Purple (K)
• (-)

  Yellow (A)
• LIA not sensitive to H2S production of Proteus
• If the organism is able to deaminate lysine, the amine converts to alpha-ketocarboxylic acid & the slant turns red
• Lysine decarboxylation


  Produces cadaverine (an alkaline end product), turning the butt purple
• No Lysine decarboxylation


  Glucose will be fermented producing acid, turning the butt yellow
• The production of H2S can mask the purple color in the butt of the tube. Because H2S production in LIA occurs only in an ALKALINE environment, it is reported as "K"
• A black precipitate indicating H2S is also positive for decarboxylation
• IMViC

  Indole, Methyl red, Voges-Proskauer, Citrate
• Indole test

  Determines the ability of the microbe to degrade tryptophan (Detects tryptophanase)

  
• Indole test principle

  Tryptophan in medium converted to indole by enzyme tryptophanase, indole will react with reagent to produce a red ring
• Methyl Red test


  Detects the ability of the organism to oxidize glucose and stabilization of high content of acid end product

  
• Methyl Red test principle

  Production of acid from dextrose, color is yellow if pH is >4.5 (negative), color is red if pH is <4.5 (positive)
• Voges-Proskauer


  Detects the ability of the organism to produce non-acidic or neutral end products, detects acetoin (acetylmethyl carbinol)
• Most Enterobacteriaciae give opposite MR & VP reactions

  
• Glucose Fermentation

  A. Mixed acid production pathway (end products: lactic, acetic, formic & succinic acid, detected by MR) B. Butylene glycol pathway (end product: acetoin, detected by VP)
• Citrate Utilization test


  Determines whether organism can utilize/ferment citrate (intermediate metabolite in the Krebs cycle) as sole source of carbon
• Malonate Test


  Determines whether organism can utilize malonate as sole source of carbon, differentiates between Escherichia (-) and Enterobacter (+)
• Decarboxylase Test

  Decarboxylation of lysine, ornithine & arginine will produce alkaline end products, detects decarboxylase enzyme
• Urease


  Determines the ability of organism to degrade urea by urease
• ONPG test

  Detects β-galactosidase, rapid test for identification of late lactose fermenters
• Phenylalanine Deaminase


  Detects PD, an enzyme that removes amino group (NH2) from an amino acid
• Gelatin Liquefaction


  Determines the ability of the organism to produce gelatinase that hydrolyze gelatin