BACT 211: LEC WEEK 14

BIOCHEMICAL CHRACTERISTICS
COLONY CHARACTERISTICS OF SOME ENTEROBACTERIACEAE 1. Carbohydrate fermentation
1. MacConkey Agar 2. Hydrogen Sulfide (H2S) production
Colorless – non lactose fermenter 3. Indole production
Pink – lactose fermenter 4. Methyl Red test
5. Voges-Proskauer test
2. SSA (Salmonella-Shigella Agar) 6. Motility test
Pink – lactose fermenter 7. Citrate Utilization test
Colorless – non lactose fermenter 8. Deamination test
Hydrogen sulfide indicator – blackening of the medium 9. Decarboxylation test
10. ONPG test
H2S producer; lactose fermenter: Citrobacter, Edwardsiella
H2S producer; non lactose fermenter: Proteus, Salmonella 1. Carbohydrate Fermentation Test Media
• TSI (Triple Sugar Iron Agar)
3. XLD (Xylose Lysine Deoxycholate) o 1% lactose, 1% sucrose, 0.1% glucose
Yellow – lactose fermenter • KIA (Kligler’s Iron Agar)
Pink – non lactose fermenter o lactose, glucose
• RDA (Russell’s Double Agar)
4. Hektoen Enteric Agar (HEA) o lactose, glucose
- Same function with SSA and XLD Indicators:
- pH indicator: Bromthymol Blue • Phenol Red – 7.3 to 7.6
Yellow / Orange – lactose fermenter • Ferrous ammonium sulfate (H2S indicator)
Blue / Green – non lactose fermenter *Yellow – utilization of carbohydrate

E. coli (colon bacillus) 1.a) Triple Sugar Iron (TSI) Agar

MAC (MacConkey Agar) – pink Description Purpose
XLD (Xylose Lysine Deoxycholate) – yellow A. Protein sources To permit bacterial growth
SSA (Salmonella-Shigella Agar) – pink Beef extract, peptone,
HEA (Hektoen Enteric Agar) – yellow/orange proteose peptone
B. Carbohydrate present For fermentation reactions
0.1% glucose
Klebsiella spp.
1% lactose
*Klebsiella pneumoniae is also known as Friedlander’s bacillus
!% sucrose
EMB (Eosin Methylene Blue) – pink
C. Indicators
XLD (Xylose Lysine Deoxycholate) – yellow Phenol red Indicates acid production due
HEA (Hektoen Enteric Agar) – yellow/orange to sugar fermentation
Acid = yellow
Enterobacter spp. Alkaline = no color change
MAC (MacConkey Agar) – pink moist
EMB (Eosin Methylene Blue) – fish eye colony Ferrous sulfate Indicates H2S production
D. Others
Proetus spp. Sodium thiosulfate Sulfur source
- Has a swarming motility (outward motility)
- Odor: burnt gun powder Triple Sugar Iron (TSI) Reactions
BAP (Blood Agar Plate) – swarming motility Two Reaction Chambers: butt-slant
Nutrient Agar – swarming motility Slant – Aerobic
- Causes UTI, gastroenteritis Butt – Anaerobic

Serratia spp.
- Red color pigment: prodigiosin
- High risk of antibiotic resistance

Yersinia enterocolitica
- Grows in MAC as lactose fermenter
- Requires highly selective media: CIN (Cefsulodin-Irgasan-
Novobiocin)
- Bull’s eye colony *Red – uninoculated
- Requires cold enrichment medium: 1-6oC for 4 hours
(minimum)
Red/Red or K/K or Alkaline/Alkaline → Non-Fermentative Group A/A + gas (aerogenic) E. coli
Red/Yellow or K/A or Alkaline/Acid → Non-Lactose Fermenter Klebsiella spp.
Yellow/Yellow or A/A or Acid/Acid → Lactose Fermenter Enterobacter spp.
H2S Indicator: Blackening of the medium Citrobacter spp.
Cracking: Gas production (Klebsiella, Enterobacter, Citrobacter)
Non-gas (Shigella)
K/K, K/A, K/A + H2S, A/A+ Gas, A/A+H2S (Possible Results)

Lactose Fermenter: A/A E. coli

without H2S Klebsiella spp.
Enterobacter spp.
Serratia spp. 2. Sulfide Indole Motility (SIM) Test
Hafnia spp. H2S production = black precipitate
Shigella sonnei Sources of Sulfur: H2S Indicator:
Yersinia enterocolytica - Sodium thiosulfate - Ferrous Sulfate
-Cystein - Ferrous citrate
-Methionine - Ferric ammonium citrate
Na2S2O3 (Sodium Thiosulfate) → H2S + FeSO4 (Iron salt) = FeS (black
Lactose Fermenter: A/A with Citrobacter spp. precipitate)
H2S Salmonella arizonae
Edwardsiella spp. 2.a. Indole Test (SIM Medium)
Tryptophan → alanine + pyruvic acid + indole (red)
Reagents:
• Kovac’s reagent
• Ehrlich’s reagent
Active chemical component:
p-dab (paradimethylaminobenzaldehyde)
Non-Lactose Fermenter: K/A Providencia spp. Result: (+) red ring formation
without H2S Morganella spp. (-) no red ring formation
Shigella spp. Indole Positive: E. coli, Klebsiella oxytoca, Proteus vulgaris,
Yersinia spp. Plesiomonas spp., Citrobacter diversus
Erwinia spp.
Arizona spp.
2.b. Sulfide Indole Motility (SIM with 0.4% agar) Test
Sulfide: (+) blackening of SIM medium
Indole: (+) red ring formation
Motility: (+) diffuse growth away from line of inoculation (test tube
brush), turbidity or cloudy
Non-Lactose Fermenter: K/A Proteus spp.
(-) growth only along inoculation line
with H2S Salmonella spp.
*All are motile EXCEPT Klebsiella spp., and Shigella spp.
Yersinia pestis: non motile at 37oC but motile at 22oC

Non-Fermentative / Oxidizers Pseudomonas spp.

K/K Burkholderia spp.
Flavobacteria spp.
Acinetobacter spp. 3. Methyl Red – Voges Proskauer Test
Medium: MR-VP Medium
Two Fermentation Pathways:
1. Mixed Acid Pathway
2. Butylene-glycol pathway
3.a. Methyl Red Test (Mixed Acid Pathway) Rapid Urease Producers:
Principle: Proteus, Providencia, Morganella
mixed acid fermentation Late or Weak Urease Producers:
glucose acid production Citrobacter, Klebsiella, Enterobacter, Yersinia, Serratia
(lactic acid, acetic, formic, succinic acid)
Reagent: Methyl Red 6. Amino Acid Deamination/Decarboxylation Test
pH indicator: Methyl Red Culture Medium: Lysine Iron Agar (LIA)
Results: pH < 4.4: red color pH Indicator: Bromcresol purple, Ferric ammonium citrate, sodium
pH > 4.5: yellow color thiosulfate
Methyl Red Positive: E. coli, Citrobacter spp., Edwardsiella Reaction: Deamination/Decarboxylation
spp., Yersinia spp., Shigella spp., Salmonella spp., Proteus Decarboxylation: anaerobe
spp., Providencia spp., Morganella spp., Deamination: aerobe

3.b. Voges Proskauer Test (Butylene-glycol Pathway)

Principle:
glucose acetoin (neutral pH)
KOH
acetyl methyl carbinol Diacetyl carbinol + guanidine
Positive: cherry red / wine red
Negative: yellow
Reagent:
a. 0.3% creatine in 40% KOH
6.a Lysine Deamination Test
b. 5% alpha naphthol in ethyl alcohol
Lysine deaminase
Lysine pyrodoxal phosphate + FeNH4 citrate +
flavin mononucleotide (burgundy red)
4. Citrate Utilization Test
Principle: use the compound sodium citrate as its sole source of
carbon and energy and ammonium salts as its nitrogen source for
growth.
- Citrate acts as the final electron acceptor
(NH4)2PO4 (ammonium phosphate) → NH3 (ammonia)
Culture Medium: Simmon Citrate Agar (SCA)
pH Indicator: Bromthymol blue (neutral pH) 6.b Amino Acid Decarboxylation Test
Product: ammonia - Measures the enzymatic ability of an organism to decarboxylate or
Results: hydrolyze an amino acid to form an amine resulting to alkaline pH
(+) Prussian Blue > 7.6 change.
(-) Green < 6.0 Lysine decarboxylase
Citrate Positive: Klebsiella spp., Enterobacter spp., Serratia spp., Lysine pyrodoxal phosphate + cadaverin +
Citrobacter spp., Providencia spp., Hafnia spp., Salmonella enteritidis CO2 (lavender)

IMViC Reaction: Indole, Methyl Red, Voges Proskauer, Citrate

Utilization
Bacteria Indole M (MR) Vi (VP) C
E. coli + + - -
Klebsiella - - + + Cadaverin – causes the medium to be alkaline
spp. Lavender – decarboxylase (+)
Enterobacter - - + + K/K K/A R/A
spp. alkaline / alkaline Alkaline slant / acid red/yellow
*To differentiate Klebsiella from Enterobacter – Motility Test purple / purple butt (glucose
*Listed bacteria are rapid lactose fermenters fermenter)
purple / yellow
5. Urease Test (-) for deamination (-) for deamination (+) for deamination
Enzyme: Urease (+) for (-) for (-) for
Substrate: Urea decarboxylation decarboxylation decarboxylation
urease
Principle: Urea (NH3) ammonia + CO2 + H20
Medium: Christensen’s Urea Agar / Broth
pH indicator: phenol red
Results: (+) fuchsia (bright) pink: >8.4
(-) yellow: < 6.8
LIA REACTIONS 7. ONPG Test
a. K/K (Alkaline/Alkaline) ONPG (o-nitrophenyl-beta-D-galactopyranoside) – determines the
- Purple/purple presence of late or slow lactose-fermenting (LLF) organisms
- (-) for deamination producing the enzyme B-galactosidase.
- (+) for decarboxylation Principle: In lactose fermentation. 2 enzymes are required: lactose
permease, beta-galactosidase.
- LF possess both enzymes
b. K/A (Alkaline/Acid) - NLF do not possess both
- Alkaline slant / Acid butt (glucose fermenter) enzymes
- Purple / yellow - LLF only possess beta-
- (-) for deamination galactosidase
- (+) for decarboxylation

8. Automated Identification
c. R/A
- Red / yellow
- (+) for deamination
- (-) for decarboxylation

• Phenylalanine Deaminase Positive:

Morganella spp., Providencia spp., Proteus spp.
Vitek system (Automated)
• Lysine Decarboxylase Positive:
Klebsiella spp., Enterobacter spp., Serratia spp., Salmonella spp.,
Edwardsiella spp.

6.c. Moeller Decarboxylase Broth

Media and reagents:
- 1% Lysine
- 1% Ornithine
- 1% Arginine
- Sterile Mineral Oil
- Important Product - Putrescine
Analytical Profile Index (Semi-Automated)
If organism has decarboxylase: it removes carboxyl group of amino
acid, forming amines (alkaline) and carbon dioxide. The alkaline
environment is indicated by the pH indicator.
*Yellow – uninoculated
*Lavender – (+)
decarboxylase
a. Lysine cadaverine (alkaline)

decarboxylase
b. Ornithine putrescine (alkaline)

dihydrolase decarboxylase
c. Arginine citrulin ornithine

Examples of organisms and their decarboxylase reactions:

- Lysine decarboxylase:
Klebsiella pneumoniae = (+) purple, alkaline
Enterobacter cloacae = (-) yellow, acidic

- Ornithine decarboxylase:
Enterobacter cloacae = (+) purple, alkaline
Klebsiella pneumoniae = (-) yellow, acidic

- Arginine decarboxylase:
Enterobacter cloacae = (+) purple, alkaline
Klebsiella pneumoniae = (-) yellow, acidic
 Gram Negative Bacilli (Glucose Fermenter, Oxidase Positive) Biotype VP Polymixin Hemolysin Agglutination
B with chicken
a. Vibrio species RBC
b. Plesiomonas species Classical Negative Susceptible Negative Negative
c. Aeromonas species Cholera
Eltor Positive Resistant Positive Positive
Family: Vibrionaceae Cholera
General Characteristics
• Gram negative curve / comma bacilli Virulence Factors of V. cholerae
• Glucose fermenter Virulence Factor Biologic Effect
• Oxidase positive Cholera toxin / Hypersecretion of electrolytes and water
• Catalase positive choleragen
• Facultative anaerobe Mucinase / Induces intestinal inflammation and
• Motile (shooting star) protease degradation of tight junctions
• Does not reduce nitrate to nitrite Pili Adherence to mucosal cells
• Halophilic (Non-halophile: V. cholerae, V.mimicus)
• Prefer aquatic habitat Mechanism of Action of Cholera Toxin

Members of Vibrio species

Species Source of Clinical Disease
Infection
V. cholerae Water, food Gastroenteritis
V. parahaemolyticus Shellfish, Gastroenteritis,
seawater wound infection,
bacteremia
V. vulnificus Shellfish, Bacteremia, wound
seawater infection, cellulitis
V. alginolyticus Seawater Wound infection,
external otitis
V. bollisae Shellfish Gastroenteritis,
wound infection,
bacteremia
V. fluvialis Seafood Gastroenteritis, Common species TCBS MAC Cholera String Kanagawa
Toxin Test Phenomenon
wound infection, V. cholerae Yellow NLF + + -
bacteremia V. parahaemolyticus Green NLF - - +
V. damsela Seawater Wound infection V. alginolyticus Yellow NLF - - -
V. metschnikovii Unknown Bacteremia V. vulnificus Green LF - - -
V. mimicus Fresh water Gastroenteritis, Stool requirement for TBCS: watery stool
wound infection, *TBCS – sucrose (yellow), non-sucrose (green)
bacteremia *MacConkey – (+) pink, (-) colorless
V. furnissii Seawater Gastroenteritis *Wagatsuma blood agar – medium used for testing Kanagawa
V. cincinnatiensis Unknown Bacteremia, Phenomenon
meningitis
• Vibrio parahaemolyticus (+) = double zone of inhibition
V. carchariae Seawater Wound (shark bite)
Biochemical Characteristics
Vibrio Cholerae Common TSI Lactose Sucrose LDC ODC ADH 6%
species NaCl
Disease: Cholera V. cholerae K/A V + + + - -
Infectious Dose: >108 CFU V. K/A - - + + - V
Two Serogroups (based on somatic antigen) parahaemolyti
cus
1. O1 (Serotypes: Ogawa (India), Inaba (Philippines), Hikojima V. alginolyticus K/A - + + V - +
(Japan)) V. vulnificus K/A + - + + - +
2. O139
CIN TSI LIA DNAse
Cholera Aeromonas Pink A/A + H2S K/A or K/K +
• Rice watery stool Plesiomonas Colorless K/A K/K -
• Severe dehydration
• Fluid and electrolyte imbalance
• Sever muscle cramping
• Anuria (inability to produce urine)
 Bile Sucrose LDC ADH ODC
Esculin
A. hydrophila - V V + -
A. caviae + + - + -
A. jandei - - + + -
A. schuberti - - + + -
A. sobria - + + + -
P. - - + + +
shigelloides

Gram Negative Bacilli (Non-glucose Fermenter / Non-Fermentative)

a. Pseudomonas
b. Burkholderia
c. Flavobacteria
d. Acinetobacter

*Colorless in MacConkey
*O/F – oxidase positive, obligate aerobe
Common denominator: K/A in TSI (all of them)

The Non-Fermenters
• Achromobacter
• Acidovorax
• Acinetobacter
• Agrobacterium
• Alcaligenes
• Burkholderia
• Stenotrophomonas
• Chryseobacterium
• Chryseomonas
• Comamonas
• Flavimonas
• Flavobacterium
• Methylobacterium
• Moraxella
• Weeksekka
• Ochrobactrum
• Oligella
• Pseudomonas
• Psychobacter
• Roseomonas
• Shewanella
• Shpingobacterium

Clinical Infections:
• Septicemia
• Meningitis
• Osteomyelitis
• Wound infections following surgery or trauma
• Nosocomial infections