Diagnostics

Gram Positive Rods

Classification

Gram positive rods Spore formers Non spore formers Aerobes & facultative aerobes Strict anaerobes Aerobes

Bacillus Clostridium Listeria

Characteristics of Spore Forming Rods

• All are large Gram positive rods • Make endospores • Mainly found in soil, water and dust • Highly diverse nutritional requirements – Use simple and complex carbon sources – Their spores allow them to resist extreme conditions

Endospore Morphology

• Location: – Terminal (a, d, e) – Subterminal (b) – Central (c, f). • Shape: – Circular (b, d) – Ellipsoid (a, c, e, f) • Spore diameter: – Non-deforming (a, b, c) – Deforming (d, e, f).

Medically Important Bacilli

• Bacillus – Mostly harmless – A few opportunistic species • Bacillus cereus & Bacillus subtilis – Food poisoning – One pathogenic species • Bacillus anthracis – Anthrax Gram positive rods Spore formers Non spore formers Aerobes & facultative aerobes Strict anaerobes Aerobes

Bacillus Clostridium Listeria

Medically Important Bacilli

• Clostridium – Several pathogenic species • Clostridium perfringens – Gas gangrene • Clostridium tetani – Tetanus • Clostridium botulinum – Botulism • Clostridium difficile – Diarrhea prolonged contraction of skeletal muscle fibers, neurotoxin produced by the bacteria Canned food that has not been sterilized properly; paralytic illness

Identification: Metabolic Tests

• Phenol red broth – Simple Carbon source: • Peptone (protein  amino acids) • Desired sugar added – pH indicator • Phenol red – Yellow acid pH – Orange neutral pH – Red alkaline pH

Phenol Red Broths

•

Carbon utilization:

– Sugar • Acid reaction (yellow) • or neutral (original) – Protein • Alkaline reaction (red)

Glucose Fermentation

• Fermentation with acid accumulation: – Glucose  pyruvate  lactic and/or acetic acid + CO 2 • Fermentation with accumulation of neutral

products

– Glucose  pyruvate 

acetoin

 2 butanediol + CO 2

Methyl Red Test

• Test for acid accumulation – Carbon Sources: Glucose and proteins – Indicator -methyl red; Added after growth • MR +: red (pH < 5.2) • MR - : Yellow (pH > 5.2) Neutral Acid

Voges-Proskauer Test

Reagents VP: butanediol +  -naphthol + KOH + O 2  acetoin VP + = red VP - = Yellow Usual results of MR/VP: MR+/VP-; MR-/VP+ MR-/VP - + Neutral Acid Acid produced No acetoin Neutral Acetoin

Negative

Urea Utilization

• Enzyme tested – Urease Positive • pH Indicator – Phenol red (turns pink)

H 2 N

C O + 2 H 2 O 

H 2 N

Urea CO 2 + H 2 O + 2 NH 3  Amino acids (NH 4 ) 2 CO 3 ammonium carbonate (alkaline)

Complex Carbon Utilization

• Too large to be transported inside • Requires exocellular enzymes for the external degradation into smaller units – Polysaccharides • Starch (amylase) – Lipids (lipase) • Tributyrin – Proteins (protease) • Casein (caseinase)

Amylase – Starch Agar

Before iodine addition After iodine addition

Caseinase – Milk Agar

Lipase – Spirit Blue

Aerobic Respiration Electron Transport Chain

2 H +

exterior

H + 2 H 2 e + Cyt

b

Cyt

o

2 e Q 2 e 2 e Fe-S 2 H + 3 H + + 3 OH

-

3 H + + 1/2 O 2 Fp H 2 O

interior

NADH + H + FADH 2 3 H 2 O

Oxidase Test

phenylenediamine • Cytochrome oxidase catalyzes the reduction of a final electron acceptor, oxygen • An artifcial e- donor, phenylenediamine , is used to reduce the cytochrome oxidase • If the enzyme is present, the colorless reagent (reduced state) will turn

blue

(oxidized state)

Catalase

We add this.

2H 2 O 2

Does bacteria make this?

catalase

2H 2 O + O 2

 Detect bubbles.

Product of respiration Damaging for DNA Add 3% H 2 O 2 to bacterial growth bubbles (O 2 ) Aerobic metabolism requires catalase

Anaerobic Respiration

2 H + Exterior 2 H + 2 e Cyt

b

2 e Q 2 e 2 e Fe-S 2 H + 3 H + + 3 OH Fp Nitrate reductase NADH + H FADH 3 H 2 2 NO 3 + 2 H + (N = +5) nitrate Final e- acceptor O +

Interior

NO 2 + H 2 O (N = +3) nitrite

Nitrate Reductase

NO 3 nitrate + 2 H + + 2 e  H 2 O + NO 2 nitrite  NO, N 2 O, NH 2 OH, NH 3 , N 2 Step 1: Test for nitrite NO 2 + sulfanilic acid and alpha naphthylamine  HNO 2 Nitrate is reduced Production of Nitrite Red Nitrate is reduced to nitrite Nitrite is reduced No Nitrite Yellow Nitrate is not reduced No Nitrite Yellow

Nitrate Reductase (Cont’d)

NO 3 nitrate + 2 H + + 2 e  H 2 O + NO 2 nitrite  NO, N 2 O, NH 2 OH, NH 3 , N 2 Step 2: Test for the presence of nitrate NO 3 + Zn (s)  NO 2 Nitrate is present Reduction to Nitrite Red Nitrate is absent Nitrite was reduced Yellow

Diagnostics

Gram Positive Cocci

Classification

Gram positive cocci

Streptococcaceae

Catalase -

Micrococcaceae

Catalase + Aerobes & facultative anaerobes Aerobes

Streptococcus Micrococcus Staphylococcus

Characteristics of Gram Positive Cocci

• All are non sporulating • Mainly found amongst the natural flora of humans and animals • Fastidious (‘picky’) nutritional requirements – Use simple carbon sources

Cellular Aggregation of Gram Positive Cocci

Micrococcus & Streptococcus Streptococcus Micrococcus Staphylococcus

Gram Positive Cocci of Medical Importance

• Micrococcaceae – Staphylococcus aureus • Causes several types of infections, food infections and toxic shock (skin and respiratory tract) – Staphylococcus epidermidis • Cause opportunistic infections (catheters with biofilms) – Staphylococcus saprophyticus • Major cause of cystitis in women (bladder infection)

Gram Positive Cocci of Medical Importance

• Streptococcaceae – Streptococcus pyogenes • Strep throat and flesh eating disease – Streptococcus agalactiae • Genital infections – Streptococcus mutans • Endocarditis – Streptococcus pneumonia • Otitis, meningitis, and pneumonia – Enterococcus spp. • Opportunistic infections

Identification: Metabolic Tests

• Microccocus Vs Staphylococcus • Oxidase test – Micrococci are + – Staphylococci are – • Bacitracin (antibiotic) – Micrococci are sensitive – Staphylococci are resistant

Identification: Metabolic Tests

• Differentitation of Staphylococcus species • Coagulase test – Coagulase positive staphylococci » S. aureus – Coagulase negative » All the other Staphylococci • Mannitol fermentation – S. aureus and some S. saprophyticus are positive – S. epidermidis is negative

S. epidermidis S. aureus

Identification: Metabolic Tests

• Differentiation of Streptoccocus species • Serological grouping according to Lancefield – Based on the type of carbohydrate in their cell wall » 8 groups (A-H and K-U) » Group A: β-hemolytic - Streptococcus pyogenes » Group B: β-hemolytic -

S. agalactiae

» Group C: α or γ hemolytic-

S. viridans

» Group D: γ –hemolytic –

Enterococcus

» Does not belong to any Lancfield group » α hemolytic-

S. pneumoniae and S. mutans

Blood Hemolysis

• Blood agar: – Discrimination according to hemolysis patterns • Alpha hemolysis – Incomplete hemolysis • Beta hemolysis- Complete hemolysis • Gamma hemolysis – No hemolysis

Identification: Metabolic Tests

• Identification of Streptoccocus pneumoniae – Bile solubilization •

Strep. pneumoniae

is positive • Other

Strep.

are negative • Identification of Enterococcus – Bile-esculin test •

Enterococcus

is positive • Other

Strep.

are negative