Biochemical Testing and Identification of Gram-Negative Bacilli

General Characteristics of Enterobacteriaceae

  • Morphology: Gram negative rods (bacilli).
  • Motility: Most are motile with peritrichous flagella.
  • Exceptions to Motility: Klebsiella and Shigella are non-motile.
  • Metabolism: Facultative anaerobes and non-spore formers.
  • Biochemical Core:
    • Ferments GLUCOSE.
    • Often produce gas during fermentation.
    • Catalase Result: Positive ($+$) for all members except Shigella dysenteriae.
    • Oxidase Result: Negative ($-$) for all members except Vibrio and Plesiomonas shigelloides.
    • Nitrate Reduction: Ability to reduce nitrate to nitrite except for Erwina and Pantoea agglomerans.

Antigenic Structure and Stability

  • K Antigen (Capsular Antigen):
    • Location: Capsule.
    • Heat Stability: Labile (heat-sensitive).
    • Examples: Salmonella spp. contain the Vi Ag (virulence antigen).
  • O Antigen (Somatic Antigen):
    • Location: Cell wall/Body.
    • Heat Stability: Stable.
    • Antibody Type: Stimulates IgM production.
  • H Antigen (Flagellar Antigen):
    • Location: Flagella.
    • Heat Stability: Labile.
    • Antibody Type: Stimulates IgG production.
    • Denaturation: Can be denatured by alcohol.

Hydrogen Sulfide (H2SH_2S) Production

  • Indicator: Blackening of the medium indicates positive H2SH_2S production.
  • Acronym "SPACED" for H2S(+)H_2S(+) Organisms:
    • S: Salmonella
    • P: Proteus
    • A: Arizona (Salmonella arizonae)
    • C: Citrobacter freundii
    • E: Edwardsiella
    • D: (Used as a placeholder in the mnemonic)
  • Medium: Detected in SIM medium or TSI.

Differential and Selective Culture Media

  • EMB (Eosin-Methylene Blue):
    • Inhibitors/Indicators: Eosin Y and methylene blue.
    • Carbohydrate (CHO): Lactose.
    • Lactose Fermenter (LF): Red/purple colonies; E. coli produces a characteristic greenish metallic sheen.
    • Non-Lactose Fermenter (NLF): Colorless colonies.
  • MAC (MacConkey Agar):
    • Inhibitors: Crystal violet and bile salts (anti-fungal and anti-Gram positive).
    • Indicator: Neutral red.
    • CHO: Lactose.
    • LF: Red/pink colonies.
    • NLF: Colorless colonies.
  • XLD (Xylose-Lysine-Desoxycholate Agar):
    • Inhibitor: Bile salt.
    • Indicator: Phenol red.
    • CHO: Xylose, lactose, and sucrose.
    • LF: Yellow colonies.
    • NLF: Red colonies (e.g., Shigella).
    • Note: H2SH_2S producers like Salmonella will show red colonies with black centers.
  • HEA (Hektoen-Enteric Agar):
    • Inhibitor: Bile salt.
    • Indicator: Bromthymol blue.
    • CHO: Salicin, lactose, and sucrose.
    • LF: Yellow/orange colonies.
    • NLF: Green/blue-green colonies.
  • DCA (Desoxycholate-Citrate Agar):
    • Inhibitor: Bile salt.
    • Indicator: Neutral red.
    • CHO: Lactose.
    • LF: Red/pink.
    • NLF: Colorless.
  • SSA (Salmonella-Shigella Agar):
    • Inhibitor: Bile salt.
    • Indicator: Neutral red.
    • CHO: Lactose.
    • Results:
      • Colorless colonies with black centers: Salmonella spp.
      • Colorless colonies without black centers: Shigella spp.
  • BSA (Bismuth-Sulfite Agar):
    • Inhibitor: Brilliant green.
    • Indicator: Bismuth sulfite.
    • CHO: Glucose.
    • Results: Black colonies are indicative of Salmonella typhi.
  • TCBS (Thiosulfate-Citrate-Bile Salt Sucrose Agar):
    • Inhibitor: Bile salt.
    • Indicator: Bromthymol blue.
    • CHO: Sucrose.
    • LF (Sucrose Fermenter): Yellow (e.g., Vibrio cholerae).
    • NLF: Green.

Triple Sugar Iron (TSI) Agar

  • Composition: Sucrose, Lactose, and Glucose in a ratio of 10:10:110:10:1.
  • Additional Components: Sodium thiosulfate (source of sulfur) and Ferrous sulfate (FeSO4FeSO_4) as the H2SH_2S indicator.
  • pH Indicator: Phenol red (Red is alkaline; Yellow is acidic).
  • Reaction Interpretations:
    • A/A (Acid Slant/Acid Butt): Glucose and lactose and/or sucrose fermented (2-3 sugars).
    • K/A (Alkaline Slant/Acid Butt): Only glucose fermented.
    • K/K (Alkaline Slant/Alkaline Butt): No sugars fermented.
    • Gas Production: Indicated by bubbles or splitting of the medium.
    • H2SH_2S Production: Blackening of the butt of the tube.

Lysine Iron Agar (LIA)

  • Principle: Detects the ability to deaminate or decarboxylate the amino acid lysine.
  • Indicator: Bromcresol purple.
  • H2SH_2S Indicator: Ferric Ammonium Citrate (produces black precipitate).
  • Reaction Sites:
    • Butt (Decarboxylation): Positive is Purple; Negative is Yellow.
    • Slant (Deamination): Positive is Red; Negative is Purple.
  • LIA Reaction Code:
    • K/K (Alkaline Slant/Alkaline Butt): Lysine Deamination ($-$), Lysine Decarboxylation ($+$).
    • K/A (Alkaline Slant/Acid Butt): Lysine Deamination ($-$), Lysine Decarboxylation ($-$).
    • R/A (Red Slant/Acid Butt): Lysine Deamination ($+$), Lysine Decarboxylation ($-$).

IMViC Tests and Individual Principles

  • Indole Test:
    • Detects: Enzyme Tryptophanase.
    • Substrate: Tryptophan.
    • Indicator/Reagents: Kovac’s reagent, Erlich’s reagent, or PDAB (paradimethylaminobenzaldehyde).
    • Positive Result: Red ring at the top.
    • Utility:
      • Distinguishes E. coli ($+$) from Klebsiella pneumoniae ($-$).
      • Speciates Proteus: P. mirabilis ($-$) vs. P. vulgaris ($+$).
  • Methyl Red (MR) Test:
    • Principle: Detects mixed acid fermentation of glucose (lactic, acetic, formic, and succinic acids).
    • Indicator: Methyl red.
    • Positive Result: Red (pH must drop to 4.44.4 or less).
    • Negative Result: Yellow (alkalinic status).
  • Voges-Proskauer (VP) Test:
    • Principle: Detects the production of acetoin (acetylmethylcarbinol), an intermediate in the butylene glycol pathway.
    • Reagents: Alpha-naphthol and 40% KOH40\% \, KOH (Barritt's or Coblentz method).
    • Positive Result: Red color due to diacetyl formation in the presence of oxygen.
    • Negative Result: Yellow/brown/salmon.
    • General Rule: Most Enterobacteriaceae are either MR($+$)/VP($-$) or MR($-$)/VP($+$).
    • VP($+$) Mnemonic "KESH": Klebsiella, Enterobacter, Serratia, Hafnia.
  • Citrate Utilization Test:
    • Medium: Simmons Citrate Agar.
    • Principle: Determines if citrate is the sole carbon source.
    • Indicator: Bromthymol blue.
    • Positive Result: Prussian blue/royal blue.
    • Negative Result: Green.

Utilization and Enzymatic Tests

  • AMA (Acetate, Malonate, Acetamide) Utilization:
    • Principle: Determines if the organism can use these as sole carbon sources.
    • Indicator: Bromthymol blue.
    • Positive: Blue; Negative: Green/Yellow.
  • Urease Test:
    • Medium: Christensen Urea broth.
    • Reaction: Urea→Urease(NH4)2CO3+NH3+CO2\text{Urea} \xrightarrow{\text{Urease}} (\text{NH}_4)_2\text{CO}_3 + \text{NH}_3 + \text{CO}_2
    • Indicator: Phenol red.
    • Positive: Pink/red; Negative: Yellow.
    • Rapid Urease ($+$) at 2 hours (PPM): Proteus, Providencia, Morganella.
    • Slow Urease ($+$): Citrobacter, Klebsiella, Enterobacter gergoviae, Yersinia, Serratia.
  • ONPG Test:
    • Detects: β\beta-galactosidase enzyme in Late Lactose Fermenters (LLF).
    • Substrate: o-Nitrophenyl-β\beta-D-galactopyranoside.
    • Positive: Yellow (production of orthonitrophenol).
    • Utility: Distinguishes Shigella sonnei (ONPG $+$) from other Shigella spp.
  • Decarboxylase Test (MOAC/LOAC):
    • Amino Acids Tested: Lysine (to Cadaverine), Ornithine (to Putrescine), Arginine (to Citrulline).
    • Indicator: Bromcresol purple.
    • Positive: Purple; Negative: Yellow.
  • PAD (Phenylalanine Deaminase):
    • Reagent: 10% FeCl310\% \, FeCl_3.
    • Positive Result: Green.
    • Positive Organisms (PPM): Proteus, Providencia, Morganella.
  • Gelatin Liquefaction Test:
    • Positive Result: Liquefaction of the medium.
    • Positive Organisms: Serratia and Proteus.

Organism Classification by Lactose Fermentation

  • Rapid Lactose Fermenters ("EKE"):
    1. Escherichia coli
    2. Klebsiella spp.
    3. Enterobacter spp.
  • Late Lactose Fermenters ("S2HYCS"):
    1. Salmonella arizonae
    2. Shigella sonnei
    3. Serratia spp.
    4. Hafnia spp.
    5. Yersinia spp.
    6. Citrobacter spp.
  • Non-Lactose Fermenters (NLF):
    • Salmonella (except S. arizonae), Shigella (except S. sonnei), Proteus, Providencia, Morganella, Edwardsiella.

Escherichia coli (E. coli)

  • Key Reactions: Indole ($+$), MR ($+$), VP ($-$), Citrate ($-$).
  • TSI: $A/A$; LIA: $K/K$ (or acidic butt variation).
  • EMB: Greenish metallic sheen.
  • MUG Test: Electric blue fluorescence ($+$ for most E. coli; $-$ for O157:H7).
  • Clinical Significance: #1 cause of UTI, #2 cause of neonatal meningitis (after Group B Streptococcus).
  • Enteropathogenic Strains:
    1. ETEC (Enterotoxigenic): "Traveller's diarrhea" or "Montezuma's revenge." Produces LT (Heat-labile) and ST (Heat-stable) toxins. Rice water stools.
    2. EIEC (Enteroinvasive): Shigella-like dysentery with blood, pus, and mucus. Slant/butt: $K/A$.
    3. EPEC (Enteropathogenic): Outbreaks in hospital nurseries ("infantile diarrhea"). Non-invasive, no toxin.
    4. EHEC (Enterohemorrhagic): Most virulent is strain O157:H7. Causes Hemolytic Uremic Syndrome (HUS). Sorbitol MacConkey (SMAC) and MUG negative.
    5. EAEC (Enteroaggregative): Acute and chronic diarrhea via aggregative adhesion fimbriae.

Klebsiella, Enterobacter, and Serratia (KESH)

  • Klebsiella:
    • Non-motile, mucoid colonies (capsulated).
    • Reactions: Indole ($-$), MR ($-$), VP ($+$), Citrate ($+$).
    • Exceptions: K. oxytoca is Indole positive.
    • Friedlander's Bacillus: Older name for K. pneumoniae.
  • Enterobacter:
    • Motile, mucoid.
    • Reactions: Indole ($-$), MR ($-$), VP ($+$), Citrate ($+$).
    • E. gergoviae is urease positive.
    • E. sakazakii produces yellow colonies at 25 ∘C25\,^\circ\text{C}.
  • Serratia:
    • Produces red pigment (Prodigiosin) at 25 ∘C25\,^\circ\text{C} (S. marcescens and S. rubidaea).
    • Produces three enzymes: DNase, Lipase, Gelatinase.
    • S. odorifera: Potato-like odor.

Salmonella and Shigella

  • Salmonella:
    • NLF, H2S(+)H_2S (+), Motile (except S. gallinarum and S. pullorum).
    • TSI: $K/A$ with H2SH_2S.
    • Widal Test: Agglutination test for diagnosis.
    • Specimens: Blood/Urine (1st week), Stool (2nd week), Bone marrow (best specimen).
    • S. typhi: Causes typhoid fever; BSA produces black colonies.
  • Shigella (Inert Bacteria):
    • NLF, Non-motile, Acetate ($-$), TSI: $K/A$.
    • Clinical: Dysentery (bloody stool with mucus).
    • Groups:
      • Group A: S. dysenteriae (Catalase $-$, ONPG $-$, Mannitol $-$)
      • Group B: S. flexneri (Catalase $+$, ONPG $-$, Mannitol $+$)
      • Group C: S. boydii (Catalase $+$, ONPG $-$, Mannitol $+$)
      • Group D: S. sonnei (Catalase $+$, ONPG $+$, Mannitol $+$, Ornithine $+$)

Proteus, Providencia, and Morganella (PPM)

  • Commonalities: PAD positive, Rapid Urease Positive.
  • Proteus:
    • High motility ("swarming" phenomenon on BAP).
    • TSI: $K/A$ with H2SH_2S.
    • P. vulgaris: Indole ($+$), OX2/OX19 source.
    • P. mirabilis: Indole ($-$), OXK source.
  • Providencia:
    • P. stuartii: Burn unit outbreaks.
    • P. rettgeri: Pathogen of the urinary tract.
  • Morganella morganii: TSI $K/A$, Ornithine ($+$), Indole ($+$).

Yersinia Species

  • General: Motile at 22 ∘C22\,^\circ\text{C}, non-motile at 35 ∘C35\,^\circ\text{C}.
  • Yersinia pestis: "Plague Bacillus." Transmitted by Xenopsylla cheopsis (rat flea). Shows "stalactite pattern" in broth and bipolar "safety pin" staining with Wayson stain.
  • Yersinia enterocolitica: #1 blood bag contaminant. Bull's eye colonies on CIN agar (Cefsulodin-Irgasan-Novobiocin). Cold enrichment at 4 ∘C4\,^\circ\text{C}.

Non-Fermentative Organisms (NFO)

  • Pseudomonas aeruginosa:
    • Oxidase ($+$), Catalase ($+$), Nitrate ($+$).
    • Odor: Sweet, grape-like, corn chips, or tortilla (due to 2-aminoacetophenone).
    • Pigments: Pyocyanin (blue), Pyoverdin (green), Pyorubin (red).
    • Differentiating Test: Growth at 42 ∘C42\,^\circ\text{C}.
    • Clinical: #1 cause of Nosocomial infections; cystic fibrosis complication; Swimmer's Ear; Hot Tub Syndrome.
  • Burkholderia:
    • B. mallei: Glander's disease (horses). Only non-motile pseudomonad.
    • B. pseudomallei: Melioidosis ("Vietnamese Time Bomb Disease"). Wrinkled colonies on Ashdown medium.
    • B. cepacia: Earthy/dirt-like odor. #2 complication in cystic fibrosis.
  • Stenotrophomonas maltophilia: Oxidase ($-$), DNase ($+$), Lavendar-green colonies.
  • Acinetobacter: #2 NFO. Oxidase ($-$), non-motile.

Vibrio, Aeromonas, and Plesiomonas

  • Vibrio:
    • Comma shaped, monotrichous flagella, Oxidase ($+$).
    • String Test positive (0.5% sodium desoxycholate0.5\% \, \text{sodium desoxycholate}).
    • Halophilic (requires NaCl) except V. cholerae and V. mimicus.
    • V. cholerae: Causes rice water stools. TCBS produces yellow colonies (sucrose ferner).
    • V. parahemolyticus: Kanagawa positive. Green colonies on TCBS.
    • V. vulnificus: Septicemia and wound infections.

Fastidious Gram-Negative Bacilli

  • Haemophilus:
    • Requires X-factor (hemin) and/or V-factor (NAD).
    • H. influenzae (Pfeiffer’s Bacillus): Satellitism around S. aureus. Type B causes meningitis in children ($<5$ years).
    • H. aegyptius (Koch-Weeks Bacillus): Contagious pink eye.
    • H. ducreyi: Chancroid (soft chancre); "School of fish" morphology.
    • ALA Test: Species that are ALA ($+$) do not require X-factor (e.g., H. parainfluenzae).
  • Bordetella:
    • B. pertussis: Agent of whooping cough. Mercury droplet/pearl-like colonies on Bordet-Gengou or Regan-Lowe agar.
  • Brucella:
    • Causes Undulant fever (zoonotic). Sub-species based on animal host: B. melitensis (goats), B. abortus (cattle/Bang's disease), B. suis (pigs), B. canis (dogs).
  • Legionella pneumophila: Requires L-cystine and iron. Identified on BCYE (Buffered Charcoal Yeast Extract) agar. Acquired from air-conditioning cooling towers.
  • Gardnerella vaginalis: Associated with bacterial vaginosis. "Clue cells" seen on Pap smear. "Whiff test" ($10\% \, KOH$) produces fishy odor.

HACEK Group (Subacute Bacterial Endocarditis)

  • H: Haemophilus aphrophilus (does not require X/V factors).
  • A: Actinobacillus (Aggregatibacter) actinomycetemcomitans (Star-like colony).
  • C: Cardiobacterium hominis (Tear drop shape, Indole positive).
  • E: Eikenella corrodens (Corrodes/pits the agar, bleach-like odor).
  • K: Kingella kingae (Twitching motility).

Questions & Discussion

  • Question: A medical technologist cultured a specimen from a suspected cystic fibrosis patient. After 24 hours of incubation, the MT noticed colonies which were spreading and flat, with serrated edges and a metallic sheen. There was a characteristic corn taco-like odor. Identify the bacteria.
    • A. Klebsiella pneumoniae
    • B. Escherichia coli
    • C. Staphylococcus aureus
    • D. Pseudomonas aeruginosa
  • Answer: D. Pseudomonas aeruginosa