MMI Lab Final: ESKAPE, Final Exam: Biochemical Tests

Enterococcus faecium

- 3rd most frequent cause of nosocomial bloodstream infections in the US

- 60% are Vancomycin resistant (VRE)

- gram positive cocci

Enterococcus faecium (relative)

Enterococcus raffinosus

Bacillus subtilis

Staphylococcus aureus

- MRSA ( methicillin resistant Staphylococcus aureus)

- gram postive cocci

Staphylococcus aureus (relative)

Staphylococcus epidermidis

Klebsiella species

- negative rod

- ESBL = Extended-Spectrum Beta-Lactamases

- Cross-resistance is growing

- Bloodstream infections from these organisms show increased mortality

Klebsiella species (relative)

Escherichia coli

Acinetobacter baumannii

- Multi-drug resistant (MDR) infections are increasing

- Usually a hospital-acquired infection

- negaitve coccobacillus

Acinetobacter baumannii (relative)

Acinetobacter baylyi

Pseudomonas aeruginosa

- Patients on ventilators or with cystic fibrosis areespecially at risk

- Negative rod

Pseudomonas aeruginosa (relative)

Pseudomonas putida

Enterobacter Species

- Health care-associated infections

- Resistance via ESBLs and carbapenemases

- negative rod

Enterobacter species (relative)

Enterobacter aerogenes

Erwinia carotovora

Selective media

-Additives suppress unwanted microbes and encourage desired microbes

- EMB, mannitol salt agar

Differential media

- Allow distinguishing of colonies of different microbes on the same plate

- alpha and beta hemolysis on blood agar; MacConkey agar, EMB, mannitol salt agar.

MAC (both selective & diff)

Selective: Bile salt/ CV

Diff: Checks if bacteria can ferment Lactose

MAC (Inhibitor)

Bile salts and Crystal Violet

-Inhibits G(+) growth

MAC Indicator

-neutral red dye

- pH > 6.8 colorless

- pH < 6.8 red (pink to red bile precipitate)

MAC substrate

Lactose

MAC description

Used to isolate and differentiate Enterobacteriaceae based on ability to ferment Lactose

lactose non-fermenters retain normal coloration

MAC poor growth

Inhibited by crystal violet/Bile

- Gram (+)

MAC pink to red growth + bile precipitate (R)

Not inhibited by CV/Bile + lactose fermentation

gram (-)

possible coliform

MAC Bright Pink

E.coli

MAC good growth and colorless colonies

not inhibited by CV/bile

- gram (-)

- noncoliform

HEA (both selective & diff)

Selective: Bile salt

Differential Components:

- Lactose, sucrose, salicin

- bromothymol blue and acid fushin

- sodium thiosulfate + ferric amonium citrate

HEA Hektoen Enteric Agar Substrates

lactose, sucrose or salicin

HEA inhibition

Bile salts

- prevents or inhibits G+ organisms

HEA used for ?

Used to isolate and differentiate Salmonella and Shigella from other enterics in patient stool samples or dairy, poultry products

HEA poor growth

Inhibited by bile or dye

- gram (+)

HEA pink/ orange growth; blue green (Bppt)

Not inhibited by bile or dye, ferments sugar

- gram (-), pi= not shigl. or samnl.

Bppt = probable salmonella

HEA blue green growth w/o black ppt

not inhibited by bile or dye

- no sugar fermentation

- gram (-)

- probable shigella

SIM Purpose

- To identify coliforms

- Useful for further ID of Enteric bacteria species

SIM

Sulfur reduction, indole production and motility

SIM (Sulfulr Reduction)

Purpose: Detects production of hydrogen sulfide (H₂S)

Key enzymes:

Cysteine desulfurase

Thiosulfate reductase

Process:

Sulfur is reduced to H₂S

H₂S reacts with ferrous ammonium sulfate in the medium

Forms black precipitate: Ferric sulfide (FeS)

SIM (Indole Production)

Purpose: Tests for ability to break down tryptophan

Key enzyme: Tryptophanase

Reaction:

Tryptophan → Pyruvate + Ammonia = indole

Detection:

- Add Kovac's reagent (contains DMAB) then reaction is red

SIM (Motility)

Purpose: Determines if bacteria are motile

Why it works:

- Soft agar (0.5%) allows movement

Observation:

M+: diffuse, radiating turbidity from stab line

M-: growth only along stab (no spreading)

Phenol Red Fermentation Test

Tests for Sugar Fermentation

Anaerobic Microbes/some aerobes

Products: acids and sometimes gases

Differentiates Enterobacteriacae from other Gram(-) rods OR Gram(+) Fermenters

Phenol Red broth (VERSIONS)

- Glucose

- Lactose

- Sucrose

- Mannitol

Phenol Red broth indicator

Phenol Red

- pH < 6.8 = yellow (acidic)

- pH > 7.4= pink/magenta (basic)

- without inoculation = 7.3 pH

Phenol Red Yellow broth/ bubbles

Notation: A/G

Sugar fermented with acid and gas products only

Phenol Red Yellow broth/ no bubbles

A/-

Sugar fermented with acid products only

Phenol Red Red broth/ no bubbles

-/-

No sugar fermentation

Phenol Red pink broth/ no bubbles

K

No sugar fermentation, peptone degraded

Urease Purpose

Urea can be hydrolyzed to ammonia (NH3) and CO2 (basic byproducts)

- Provides nitrogen in usable form (ammonia)

- Urease is activated by bacteria when usable N is absent or urea is present

- Pathogenic Urease + bacteria:

Proteus (+)

H. Pylori (+)

Urease Results

Indicator = Phenol red

Pink color in 24 hr = rapid urease +

Orange/ yellow = -

TSIA (Triple Sugar Iron Agar)

Differential Medium ONLY

Differentiates organisms based on the ability to reduce sulfur and ferment carbohydrates (glucose, lactose, sucrose).

Yellow = acidic

Pink = basic

Differentiates Enterobacteriaceae from other Gram (-) rods like Pseudomonas aeruginosa

TSIA Yellow butt / yellow slant

Symbol: A/A

Glucose and lactose and/or sucrose fermentation with acid accumulation in slant and butt.

TSIA Red Slant/ yellow butt

Symbol: K/A

Glucose fermentation with acid production. Proteins catabolized aerobically (in the start) with alkaline products (reversion)

TSIA Red slant/ red butt

Symbol: K/K

No fermentation. Peptone catabolized aerobically and anaerobically with alkaline products. Not from Enterobacteriaceae

TSIA red slant/ no change in butt

Symbol: K/NC

No fermentation. Peptone catabolized aerobically with alkaline products. Not from Enterobacteriaceae

TSIA black ppt

Symbol: H2S

Sulfur reduction. (An acid condition, from fermentation of glucose or lactose, exists in the but even if the yellow colors is obscured by the black ppt)

TSIA Cracks or lifting

gas production

nitrate

Anaerobic respiration doesn't use O2 as final electron acceptor uses Nitrate

Enzyme Nitrate Reductase reduces Nitrate to Nitrite

Differentiates Enterobacteriaceae from other Gram(-) rods

Tests for: presence of NITROGEN REDUCTASES in bacteria and what they reduce nitrate into

Nitrate Test 1 Bubbles

Denitrification microbe is NOT a fermenter reduced nitrate to N2

Test DONE

Nitrate Test 2 (after no bubbles)

Add Reagent A and B

If tube turns red; microbe reduces nitrate to nitrite

Test Done

Nitrate Test 3 (after Reagant A, B)

Add zinc powder

If it turns red; microbe does NOT reduce nitrate

TEST done

Nitrate Test 3 (after Reagent A, B)

After adding power, still not red

microbe reduces nitrate not nitrite (NH4+, NO, N2O)

Test Done

BEA (selective and differential)

Selective: Bile salt

- inhibits most gram (+)

Diff: Esculin, ferric citrate.

BEA

Many bacteria have esculinase, but few can hydrolyze it in the presence of bile

Used for isolation and presumptive ID of BE+Enterococci (Enterococcus faecalis and E.faecium)

Or to differentiate Group D Streptococci (S. bovis, S. equinus, S. gallolyticus) from non-Group D streptococci Or to distinguish between the genera Enterobacter, Klebsiella, and Serratia (BE+) from other genera in Enterobacteriaceae

BEA Results

Positive = black ppt

No blackening = - result

MSA (selective & differential)

Selective: 7.5% Salt

Diff: Mannitol, phenol red

MSA

Used for isolation and differentiation of Staphylococcus aureus (yellow) from other Staphylococcus species

MSA Substrate (makes it differential)

Mannitol (sugar)

Indicator = phenol red

MSA poor growth

Inhibited by NaCl

- Not staph

MSA yellow/halo

Not inhibited by NaCl

- possible stap aureus

MSA red growth/ halo

Not inhibited by NaCl

- Staphylococcus other than S. aureus