BACT211: Clinical Bacteriology Week 5 - Staphylococci

Characteristics of Staphylococci

• Staphylococci belong to the family Staphylococcaceae
• They are catalase positive (+) and coagulase positive (+) or negative (-)
• They are gram positive (+) cocci that exist singly, in pairs, and in clusters
• They resemble the family Micrococcaceae and genus Micrococcus
• Micrococci are catalase positive (+), coagulase negative (-), and exist in pairs, tetrads, and irregular clusters

Differentiation between Staphylococci and Micrococci

• Modified oxidase (Microdase) test: Staphylococci are negative (-), Micrococci are positive (+)
• Anaerobic acid production from glucose: Staphylococci are positive (+), Micrococci are negative (-) except for M. kristinae and M. varians
• Anaerobic acid production from glycerol in the presence of erythromycin: Staphylococci are positive (+), Micrococci are negative (-)
• Growth on Furoxone-Tween 80-oil red agar: Staphylococci are negative (-), Micrococci are positive (+)
• Resistance to bacitracin (0.04 U): Staphylococci are resistant (R), Micrococci are sensitive (S)
• Resistance to lysostaphin (200 ug/mL): Staphylococci are sensitive (S), Micrococci are resistant (R)

Staphylococci Pathology

• Staphylococci can cause cutaneous infections such as folliculitis, furuncles, and carbuncles
• They can also cause food poisoning, scalded skin syndrome, toxic shock syndrome, and toxic epidermal necrolysis
• S. aureus can cause bullous impetigo, which differs from streptococcal nonbullous impetigo in that staphylococcal pustules are large and surrounded by a small zone of erythema
• Other staphylococcal species can cause nosocomial infections, wound infections, septicemia, UTIs, and native valve infections

Virulence Factors of Staphylococcus aureus

• Enterotoxins (Groups A-E and G-J) cause staphylococcal food poisoning
• Toxic shock syndrome toxin-1 (TSST-1) causes toxic shock syndrome
• Exfoliative toxins (A and B) cause scalded skin syndrome and bullous impetigo
• Cytolytic toxins (α, β, γ, and δ hemolysins) affect red blood cells and leukocytes
• Protein A binds to the Fc portion of immunoglobulin G (IgG) and blocks phagocytosis
• Enzymes such as coagulase, protease, hyaluronidase, and lipase aid in bacterial spread and infection

Laboratory Diagnosis of Staphylococci

• Culture media used include SBA, MSA, CAN, PEA, and CHROMagar Staph aureus
• Incubation at 35-37°C for 18-24 hours
• Macroscopic examination reveals colonies that are round, smooth, white, and creamy for S. aureus, and small-to-medium-sized, nonhemolytic, gray-to-white colonies for S. epidermidis
• Microscopic examination reveals gram-positive (+) cocci in singly, in pairs, and in clusters
• Mannitol salt agar (MSA) is selective for S. aureus, which ferments mannitol and forms colonies surrounded by a yellow halo### Staphylococci Characteristics
• Produce hemolytic zones around colonies on Sheep's Blood Agar (SBA)
• Gram-positive cocci, often in clusters, with polymorphonuclear cells
• Catalase test: positive, with bubble formation, indicating breakdown of hydrogen peroxide into oxygen and water

Microdase Test (Modified Oxidase Test)

• Principle: oxidase enzyme reacts with oxidase reagent and cytochrome C to form indophenol, a colored compound
• Result: positive, with blue to purple-blue color, indicating micrococci and staphylococci

Biochemical Tests

• Bacitracin Test: determines effect of bacitracin on an organism, with positive result indicating presence of zone of inhibition around the disk
• Oxidation-Fermentation (O/F) Reactions: determines whether an organism uses carbohydrate substrates to produce acid byproducts, with positive result indicating acid production
• Coagulase Test: differentiates Staphylococcus aureus from CoNS by detecting enzyme coagulase, with positive result indicating fibrin clot formation

Coagulase Enzyme

• 2 forms: bound coagulase (clumping factor) and free coagulase (staphylocoagulase)
• Bound coagulase: reacts directly with fibrinogen in plasma, resulting in fibrin clot formation
• Free coagulase: extracellular protein enzyme, reacts with coagulase-reacting factor (CRF) to form coagulase-CRF complex, resulting in fibrin clot formation

Pyrrolidonyl Arylamidase (PYR) Test

• Principle: differentiates S.aureus from other CoNS by presence of enzyme L-pyrroglutamylaminopeptidase
• Result: positive, with bright red color, indicating S.lugdunensis, S.intermedius, and S.schleiferi

Methicillin-Resistant Staphylococcus aureus (MRSA)

• Penicillin-resistant (β-lactam) strains of S.aureus due to presence of mecA gene
• Requires penicillinase-resistant penicillins, such as nafcillin, oxacillin, or cefoxitin
• Types: CA-MRSA, HA-MRSA, MRSA, MRSE

Vancomycin-Resistant Staphylococcus aureus (VRSA)

• VRSA: vancomycin-resistant S.aureus
• VISA: vancomycin-intermediate S.aureus
• TOC: Vancomycin

Macrolide Resistance

• Resistance to clindamycin
• Modified double disk diffusion test (D-zone test)