Staphylococcus genus: characteristics and classification

• Staphylococcus is a genus of Gram-positive bacteria.

Classification of Bacteria

• Domain: Bacteria
• Phylum: Firmicutes
• Class: Bacilli
• Order: Bacillales
• Family: Staphylococcaceae
• Genus: Staphylococcus

General Characteristics

• Morphology: Gram-positive cocci arranged singly, in pairs, or in clusters like bunches of grapes
• Non-sporulated
• May be capsulated
• Non-motile
• Isolation: Facultative anaerobic
• Grows well on the surface of ordinary media
• Most strains grow in the presence of 15% NaCl or 40% bile
• Catalase +ve
• Oxidase -ve
• Sensitive to lysis by lysostaphin endopeptidase
• Resistant to lysozyme

Lysostaphin Endopeptidase

• First isolated from a culture of Staphylococcus simulans
• It can function as a bacteriocin (antimicrobial) against Staphylococcus aureus.
• An antibacterial enzyme capable of cleaving the crosslinking pentaglycine bridges found in the cell wall peptidoglycan of certain staphylococci
• S. aureus cell walls contain high proportions of pentaglycine, making lysostaphin a highly effective agent against both actively growing and quiescent bacteria

Staphylococcus Cell Wall

• Composed of 3 layers: capsule, specific protein A, and peptidoglycan
• Capsule: A microcapsule seen only by electron microscope and present in S. aureus.
• Peptidoglycan: Contains L-lysine and teichoic acids that may be ribitol (S. aureus) or glycerol (S. epidermidis).
  • Teichoic acids functions:
    • Protection against the action of lysozymes
    • Sites for bacteriophage adhesion
• Specific Protein A (SPA):
  • The antigenic part of the cell wall, constituting 40-60% of the cell wall
  • Binds nonspecifically with the Fc portion of immunoglobulins (IgG), termed a pseudo-immune reaction
  • True reaction is the binding with the Fab portion of immunoglobulins, which leads to:
    • Prevention of Fc-mediated opsonization
    • Complement activation contributed by a vigorous inflammatory reaction
    • Septic shock syndrome as a result of massive complement activation
  • Shows a strong correlation with coagulase production
  • Importance:
    • Diagnosis of organisms unable to make agglutination and precipitation, such as ILT virus
    • Identification of toxins, such as C. perfringens toxins
  • Detection of SPA is done by:
    • Agar gel immunodiffusion test
    • Hemagglutination test

Classification According to Clinical Syndrome

• Well-documented clinically important species:
  • S. aureus is the most pathogenic
  • S. epidermidis is the less pathogenic
  • S. saprophyticus is the least pathogenic
• Probably clinically important species:
  • Isolated from infected cases, such as pneumonia and hepatitis (e.g., S. simulans and S. hominis)

Bergy's Manual Classification

• Based on coagulase test and novobiocin sensitivity.
  • Coagulase positive and novobiocin sensitive: S. aureus group (S. aureus, S. intermedius, S. hyicus)
  • Coagulase negative and novobiocin sensitive: S. epidermidis group (S. simulans, S. warneri, S. epidermidis, S. hominis)
  • Coagulase negative and novobiocin resistant: S. saprophyticus group (S. gallinarum, S. xylosus, S. saprophyticus and S. cohnii)

Differences Among Species

Pathogenicity

• Staphylococci are widely distributed in nature as opportunistic organisms

S. aureus

• Found in the anterior nares, skin, and upper respiratory tract (potential pathogen)
• Botryomycosis in the stump of the spermatic cord in horses
• Mastitis and suppurative lesions in cattle
• Superficial infections as pyoderma, folliculitis, and abscesses in humans
• Deep infections as osteomyelitis, bacteremia, septicemia, and pneumonia in humans
• Toxin diseases include exfoliatins (scalded skin syndrome and scarlet fever), enterotoxins (food poisoning), and TSST-1 (toxic shock syndrome)

S. epidermidis

• Stich abscesses, endocarditis, and human acne pustules

S. saprophyticus

• Nonpathogenic, but some strains cause urinary tract infection

Products of Staphylococcus aureus

• Hemolysins or Lysins: alpha-hemolysin, beta-hemolysin, gamma-hemolysin, and delta-hemolysin
• Enterotoxins: A, B, C, D, E, and F
• Toxic Shock Syndrome Toxin-1 (TSST-1)
• Leukocidin
• Epidermolytic toxins (Exfoliatins)
• Staphylokinase (Fibrinolysin or Plasmin)
• Coagulase (Free and Bound)
• Deoxyribonuclease (DNase)
• Phosphatase
• Penicillinase
• Fatty Acid Modifying Enzyme (FAME)
• Hyaluronidase

Exotoxins - Hemolysins or Lysins

• α-Haemolysin: Arrhenius toxin (loses activity at 70°C, retains activity at 100°C due to dissociation) , Coagulation prevent it's toxic effect , Causes dermonecrosis , Serves as virulence index , Systemic release causes septic shock. Detection by culturing S.aureus on rabbit blood agar.

Enterotoxins

• Production: 30-50% of S. aureus strains produce enterotoxins, High carbon dioxide tension (30%)
• Properties: Protein, heat stable, Resist boiling for 30 min, Resist pepsin and trypsin
• Effect: Ingestion/injection stimulates CNS (vomiting), GIT receptors cause vomition and diarrhea (food poisoning) , Detection: agar gel and ELISA.
• Detection of S. aureus in food is useless as cooking heat is sufficient to kill.

Toxic Shock Syndrome Toxin-1 (TSST-1)

• A pyrogenic exotoxin, causes Toxic shock syndrome Superantigens that bind directly to MHC-II on APC and Vβ element of T-cell, Non-specifically stimulates T-cells to release cytokines

Leukocidin

• A non-hemolytic toxin that kills polymorphic leukocytes and macrophages
• Two methods used to study leucocidal activity: Neisser Wechsberg and Panton Valentine

Epidermolytic Toxins (Exfoliatins)

• Types A (chromosomal gene) and B (plasmid), key in the pathogenesis of staphylococcal skin infections
• Causes scalded skin syndrome and extensive separation of the epidermis

Enzymes - Staphylokinase (Fibrinolysin or Plasmin)

• Action: Digests fibrin, Dissolves recent clot, Activator of plasminogen
• Detection: Inoculation of S. aureus on heated 10% human agar (opaque) à lysis of fibrin

Enzymes - Coagulase

• Action: Deposition of fibrin > produce clot > prevent phagocytic cell to engulf S. aureus
• Its a marker of pathogenic strains of S. aureus
• Detection: Cultivate in phosphate agar, expose to ammonium vapour --> Pink

Enzymes - Penicillinase

• Effect: Breaks Beta ring, inactivates penicillin

Enzymes - Fatty Acid

• Effect: modify anti-bacterial lipids to prolong bacterial survival in abscesses

Enzymes - Deoxyribonuclease (D Nase)

• a relation between Dnase activity and coagulase activity
• Coagulase +ve = Dnase +ve
• Dnase +ve = coagulase +ve
• But some coagulase -ve --> Dnase +ve Its detection: Dnase test medium, incubate 18 hrs --> flood plate with Hcl - +ve clear zone

Antibiotic Substances

• Staphylococcin
• Lysostaphin

N.B Differences Between Types of Coagulase

Isolation of S. Aureus

• Media used: general media (nutrient agar, blood agar, milk agar) selective media (mannitol salt agar, polymyxin agar, Baird Parker agar, Staphylococcus medium No 110)
• Oxygen Requirement: Facultative Anaerobic
• Optimum Temperature: 37°C.
• Incubation: 1-2 days

Identification - Culture

• selective media, S. aureus ferments mannitol and turns agar yellow
• black colonies and yellow hallow with mannitol

Identification - Colony Morphology

• Round, convex with diameter 1-4mm large colonies

Identification - Pigment Production

• Varies from white to orange and stimulates pigment production
• Golden yellow

Identification - Haemolysis

• S.aureus shows B haemolysis

Morphology

• Gram-positive cocci, Arranged in clusters
• Small to large in size
• Non-sporulated
• Non-motile
• May be Capsulated

To Differentiate S. aureus from other species

• Coagulase Test (+ve)
• Fibrinolysin (+ve)
• Mannitol Test (+ve)
• Novobiocin (sensitive)

To Differentiate Micrococcus from Staphlococcus