BACT211

CLINICAL BACTERIOLOGY
BATCH
2021

COLLEGE OF MEDICAL LABORATORY SCIENCE-OUR LADY OF FATIMA UNIVERSITY (LAGUNA CAMPUS)

Staphylococci
Characteristics
•Family Staphylococcaceae
•Catalase (+)
•Coagulase (+) & some are Coagulase (-) CoNS
•Gram (+) cocci in singly, in pairs and in clusters
•Resembles the family Micrococcaceae ; genus Micrococcus Folliculitis:
- Catalase (+) mild inflammation of a hair follicle or oil gland; the infected area is raised and red
- Coagulase (-) Furuncles
- Gram (+) cocci in pairs, tetrads, and, ultimately, irregular clusters (boils), extension of folliculitis, are large, raised, superficial abscesses
- Found in environment and human skin •Rothia mucilaginosa
Carbuncles
- Colonies: yellow pigment •Aerococcus
occur when larger, more invasive lesions develop from multiple furuncles, which
•Alloiococcus otitis
can progress into deeper tissues
- with fever and chills, indicating systemic spread
Bullous impetigo
caused by S. aureus differs from streptococcal non-bullous impetigo in that
staphylococcal pustules are larger and surrounded by a small zone of erythema

Staphylococcus aureus
Six Virulence factors:
1. Enterotoxins
• Groups A-E & G-J Serologically identified
• Staphylococcal food poisoning A,B ,D Reheating contaminated food
does not prevent disease
• TSS B, C , G, I
• Staphylococcal pseudomembranous enterocolitis B
• Heat stable exotoxins (100° C for 30 mins)
• Interacting with TSST-1: interact with T cells activating an aggressive, over
reactive immune response
2. Toxic Shock Syndrome Toxin-1
• TSS
• previously known as enterotoxin F
• Super-antigen stimulating T-cell proliferation
• production of a large amount of cytokines for the symptoms
• low concentrations: causes leakage by endothelial cells
• higher concentrations: cytotoxic

3. Exfoliative Toxin
• Epidermolytic toxin
• Staphylococcal SSS Ritter disease
• Bullous impetigo

4. Cytolytic Toxin
• extracellular proteins that affect red blood cells and leukocytes
Staphylococci • Lysins & leukocidins
• Staphle meaning “bunches of grapes” • S. aureus: α, β, γ, δ hemolysins
• nonmotile, non–spore-forming, and aerobic or facultatively anaerobic EXCEP - α-hemolysin: lyse RBC, damage platelets and macrophages and causes
S. saccharolyticus (obligate anaerobe) severe tissue damage
• Normal flora of skin and mucous membranes of human & animals - β-hemolysin: also known as Sphingomyelinase C or 'hot-cold lysin"
• Colonies: medium sized (4 to 8 mm) and appear cream-colored, white or rarely : acts on sphingomyelin in the plasma membrane of erythrocytes
enhanced hemolytic activity on
light gold, and “buttery-looking” : act in CAMP test (Christie, Atkins, and Munch-Petersen)
incubation at 37° C and subsequent
• Fastidious strains requirements: CO2, hemin, or menadione with at least 48 hrs
exposure to cold (4° C)
of incubation

4. Cytolytic Toxin
• δ-hemolysin: less toxic than α-hemolysin or β-hemolysin
• γ-hemolysin: (PVL) Panton-Valentine leukocidin
Hemin- Factor X
• Staphylococcal leucocidin
Menadione – Vitamin K3 • (PVL) Panton-Valentine leukocidin
• exotoxin lethal to PMN
• suppresses phagocytosis
• associated with severe cutaneous infections and necrotizing pneumonia
• often associated with community-acquired staphylococcal infections

5. Enzymes
• Coagulase, protease, hyaluronidase & lipase
• Staphylocoagulase
- S. aureus
• Hyaluronidase
- S. aureus
• hydrolyzes hyaluronic acid present in the intracellular ground substance that
makes up connective tissues, permitting the spread of bacteria during infection
• Coagulase, protease, hyaluronidase & lipase
• Lipase
- by both coagulase (+) and CoNS
- act on lipids present on the surface of the skin

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 BACT211
CLINICAL BACTERIOLOGY
BATCH
2021

COLLEGE OF MEDICAL LABORATORY SCIENCE-OUR LADY OF FATIMA UNIVERSITY (LAGUNA CAMPUS)

6. Protein A • Bacitracin test

• Cellular components in cell wall of S. aureus - Principle: determine the effect of a small amount of bacitracin (0.04U) on an
• bind the Fc portion of immunoglobulin G (IgG) organism
• block phagocytosis and inhibit action of IgG •Result:
(+) presence of zone of inhibition around the disk
Workflow (-) no zone of inhibition
Specimen collection
• From the site of infection with aseptic technique
• No special procedures
• Aspirates: ideal sample
• Swabs: less satisfactory for both culture and smear results
SBA – Sheep’s Blood Agar
Direct Microscopic Examination MSA – Mannitol Salt Agar
• Gram (+) cocci with PMN cells CAN – Colistin and Nalidixic Agar
PEA – Phenylethyl Alcohol Agar
Culture
•SBA, MSA, CNA, PEA, CHROMagar Staph aureus
•18 to 24 hours of incubation at 35° C to 37° C

Macroscopic examination • Oxidation-Fermentation (O/F) reactions

• Colonies: - Principle: determine whether an organism uses carbohydrate substrates to
- S. aureus: round, smooth, white, creamy colonies on SBA produce acid byproducts using OF glucose medium
- S. epidermidis: small- to medium-sized, nonhemolytic, gray-to-white colonies •Result: glucose, xylose, mannitol, lactose, sucrose, and maltose
Microscopic examination (+) yellow medium; acid production staphylococci
• Gram (+) cocci in singly, in pairs and in clusters (-) no change in color; no acid production micrococci S. saprophyticus
S. auricularis
except
S. hominis
M. kristinae and
Mannitol Salt Agar S. xylosus
M. varians
S. cohnii

• Oxidation-Fermentation (O/F) reactions

• Fermentation
- GLUCOSE → PYRUVIC ACID; end product
- single acid (homolactic acid fermenters)
- mixed acids (lactic acid, propionic acid & succinic acid)
• Oxidation
- GLUCOSE → PYRUVIC ACID → CO2
- requires oxygen (aerobic respiration)
- inorganic molecule (anaerobic respiration)
• 7.5 % NaCl (mahon) or 10% baileys in MSA: selective for S. aureus
• Ferment mannitol; phenol red as pH indicator
• Forming: colonies surrounded by yellow halo
S. Saprophyticus: may also ferment mannitol; may resemble colonies of S. aureus
•may produce hemolytic zones around the colonies

Workflow
Biochemical Tests
• Catalase
- Principle: catalase mediates the breakdown of hydrogen peroxide (30% H2O2)
into oxygen and water
•Result:
(+) bubble formation staphylococci & micrococci
(-) no or few bubble formation

• Microdase test (aka Modified Oxidase Test)

- Principle: oxidase enzyme reacts with the oxidase reagent and cytochrome C to
form the colored compound, indophenol after 2 mins
•Result:
(+) development of blue to purple-blue color micrococci
(-) no color change staphylococci

• If results are suggesting that the genus is staphylococci, proceed to ......

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 BACT211
CLINICAL BACTERIOLOGY
BATCH
2021

COLLEGE OF MEDICAL LABORATORY SCIENCE-OUR LADY OF FATIMA UNIVERSITY (LAGUNA CAMPUS)

• Coagulase Test
- Principle: used to differentiate Staphylococcus aureusfrom CoNS by detecting
enzyme coagulase
• Result:
(+) fibrin clot S. aureus
(-) no fibrin clot CoNS

• Coagulase Test
• 2 forms of coagulase enzyme:
• Bound coagulase/ clumping factor Slide Coagulase Test
- bound to the bacterial cell wall and reacts directly with fibrinogen in plasma
- alteration of fibrinogen precipitates on the staphylococcal cell
- Fibrin clot (clumping of cells) (+) S. aureus, S. lugdunensis and S. schleiferi

•Free coagulase/ Staphylocoagulase Tube Coagulase Test

• extracellular protein enzyme (free coagulase) + CRF (coagulase-reacting factor)
• coagulase-CRF complex + fibrinogen
• fibrin clot possibility of autolysis due to fibrinolysin

•Pyrrolidonyl Arylamidase (PYR) Test

Principle: differentiate S. aureusfrom other CoNS by the presence of the enzyme
L- pyrroglutamylaminopeptidase
• L-pyrroglutamylaminopeptidase → L-pyrrolidonyl-β-naphthylamide/PYR
(substrate) = β-naphthylamine
• β-naphthylamine + N, N-methylaminocinnamaldehyde/p
dimethylaminocinnamaldehyde (reagent)
•Result:
(+) Bright red color S. lugdunensis, S. intermedius, S. schleiferi
(-) No color change or an orange color S. aureus

• Vogue-Proskauer (VP) test

•Principle: determine the ability of some organisms to produce neutral end
products from glucose fermentation acetyl-methylcarbinol or acetoin
•Result:
(+) Red color S. aureus
(-) Yellow color S. intermedius, S. lugdunensis, S. haemolyticus& S. schleiferi

• Novobiocin Susceptibility Test

Principle: determine the effect of a 5-μg novobiocin disk on an organism
•Result:
(+) presence of zone of inhibition around the disk other CoNS
(-) no zone of inhibition S. saprophyticus

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