Staphylococcaceae : Staphylococcus aureus and Clinical Implications

Classification of Gram Positive Cocci

a) Micrococcaceae Family

• Staphylococcus
• Micrococcus

b) Streptococcaceae Family

• Streptococcus
• Pneumococcus
• Enterococcus

Micrococcaceae Family

Difference between Staphylococcus and Micrococcus

Note:

• Hugh and Leifson medium used to assess glucose utilization.
• Bromothymol blue is the indicator.

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Staphylococcus aureus

• Bunch of grapes appearance.
• Non-motile.
• Non-sporing.
• Catalase positive.
• Oxidase negative.
• Produce golden pigment.

Virulence Factors

• Cell Wall
  • Peptidoglycan.
  • Teichoic acid—for attachment.
• Cell Surface
  • Protein A.
    • Anti-complementary.
    • Anti-phagocytic.
    • Responsible for co-agglutination.

Co-agglutination Test

• Used to detect soluble antigens.
• COWAN I strain is used for Staphylococcus aureus.
• Protein A binds to the Fc segment of IgG.
• Fab region left free.
• Antigen can get attached and form clumps.

Toxins

• Hemolysins
  • Alpha-hemolysins.
    • Inactivated at 70 degrees and activated again at 100 degrees.
    • Lyse sheep and human red blood cells.
  • Beta-hemolysins or sphingomyelinase.
    • Hot-cold phenomenon, i.e., starts working at 37°C and completes at 4°C.
    • Lyse sheep red blood cells only contain a high content of sphingomyelin.
  • Gamma-hemolysins.
    • Lyse sheep and human red blood cells.
    • Synergohymenotropic toxin.
  • Delta-hemolysins.
    • Lyse sheep and human red blood cells.
• Panton-Valentine (PV) Toxin
  • Secreted by Methicillin-resistant Staph. aureus (MRSA).
  • Synergohymenotropic toxin with gamma-hemolysin.
• Epidermolytic/Exfoliative Toxin
  • Staphylococcal scalded skin syndrome (SSSS).
    • Site of action for toxin is Stratum granulosum (desmoglein 1).
    • Ritter's disease, i.e., SSSS in children.
    • Toxic Epidermal Necrolysis (TEN).
• Enterotoxin
  • A-I, not F, R-T, V.
  • Enterotoxin A is the most common.
  • Heat stable.
  • Causes food poisoning.
  • Vagomimetic action leading to emesis.

Differential Diagnosis of Food Poisoning

• The incubation period of food poisoning is 1-6 hours, i.e., vomiting occurs 1-6 hours after food consumption.
• Food poisoning may be due to:
  • Staphylococcus aureus (Milk, meat).
  • Bacillus cereus (Chinese fried rice).

Enterotoxin F or TSST

• Toxic shock syndrome toxin.
• Associated with long-term tampon usage.
• Superantigen.

Criteria for Toxic Shock Syndrome

• Fever > 102°F.
• Hypotension with systolic blood pressure < 90 mmHg.
• Maculopapular rash.
• Involvement of any 3 of the following organs:
  • GIT -> nausea, vomiting.
  • Kidney -> serum creatinine levels 2 times the norm.
  • Liver -> SGOT and SGPT levels 2 times the norm.
  • CNS -> confusion.
  • Muscle -> creatine phosphokinase (CPK) level 2 times the normal.
  • Mucous membranes -> hyperemia.
  • Coagulopathy -> platelet count < 100,000.

Enzymes

• Thermonuclease.
• DNAase.
• Phosphatase.
• Catalase.
• Coagulase.

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Clinical Features

• Skin and soft tissue infections
  • Abscess.
  • Cellulitis.
  • Furuncles.
  • Botryomycosis.

• Osteomyelitis.
• Food poisoning.
• Toxic shock syndrome.
• Toxic epidermal necrolysis.
• Pneumonia
  • Hospital-acquired.
  • Ventilator-acquired.
• Pneumatocele.
• Acute infective endocarditis.
• Infective arthritis.
• Necrotizing fasciitis.
• Sepsis.

Staphylococcus aureus is the most common cause of:

• Osteomyelitis.
• Hospital-acquired pneumonia.
• Ventilator-acquired pneumonia.
• Acute infective endocarditis.
• Native valve endocarditis.

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Diagnosis

1. Microscopy

• Gram positive.
• Purple branch of grapes.

2. Culture

• Nutrient Agar
  • Golden color in nutrient agar.
  • Non-diffusible β-carotene staphyloxanthin pigment produced at 22 °C.

• Blood Agar
  • Pin-head colonies with narrow zone beta-hemolysis.
• Selective Media
  • S. aureus can survive in NaCl 7-10%.
  • Mannitol salt agar (MSA).
  • Ludlam's agar.
  • Salt milk agar.

3. Mannitol Salt Agar

• Salt presence makes it a selective medium.
• Mannitol presence helps in determining whether bacteria's mannitol fermentation is positive or negative, making it a differential medium.
• Phenol red is the indicator.
• Mannitol fermentation in S. aureus causes color change to yellow.

4. Biochemical Tests

• Catalase positive.
• Coagulase positive.

Catalase Test

• Culture + H₂O₂ + catalase --> H₂O + O₂ --> Bubbles.

Catalase Positive Bacteria:

• Staphylococcus aureus.
• Serratia spp.
• Pseudomonas spp.
• Escherichia coli.
• Enterobacteria.
• Bacillus anthracis.
• Neisseria gonorrhoeae.
• Neisseria meningitidis.
• Mycobacterium tuberculosis.
• Micrococcus spp.

Catalase Positive Fungi:

• Aspergillus spp.
• Candida spp.
• Cryptococcus spp.

Coagulase Test

• Slide Coagulase (Bound Coagulase): Positive in:
  • Staphylococcus aureus.
  • Staphylococcus intermedius.
  • Staphylococcus hyicus.
  • Staphylococcus lugdunensis.
• Tube Coagulase (Free Coagulase): Positive in:
  • Staphylococcus aureus.
  • Staphylococcus intermedius.
  • Staphylococcus hyicus.
  • Staphylococcus schleiferi.

• Rabbit/human plasma + culture broth (containing coagulase).
• Plasma contains coagulase-reacting factors (CRF).
• A CRF-coagulase complex formed, activating fibrinogen and clotting plasma.

Slide Coagulase Test / Clumping Factor

• Coagulase is bound to the cell wall.
• Reacts directly with fibrinogen in plasma, leading to precipitation on the cell wall and clumps of cocci.

5. Phage Typing

• Makes use of bacteriophages.
• Phage 80/81A is associated with Staphylococcus infections that lead to outbreaks.
• Phage III is associated with MRSA.

6. Genotypic Typing

• Pulse field gel electrophoresis (PFGE).

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Treatment

• Penicillin/β-lactams are the drug of choice.
  • Mechanism of action: Bind to penicillin-binding proteins (PBPs) 1, 2, 3, and 4 that are also known as transpeptidases and break their bonds.
• Resistance: β-lactamase production regulated by BLA genes and plasmids.
  • Transfer of resistances mediated by transduction > conjugation.
• Methicillin
  • Given in penicillin resistance.
  • Resistance by chromosomal-mediated MecA gene leading to MRSA with PBP altered to PBP2a that has a lower affinity for methicillin.

MRSA

Diagnosis of MRSA

• The best method is the dilution test.
• Minimum inhibitory concentration: minimum amount of antibiotic required to kill bacteria.
• MIC for cefoxitin ≥ 8 μg/ml indicates resistance.
• MIC for oxacillin ≥ 4 μg/ml indicates resistance.
• Cefoxitin disc diffusion agar/oxacillin.
  • Cefoxitin is a better determinant than oxacillin.
• Latex agglutination test for PBP2a.
• ELISA for MecA.
• PCR for MecA.

MRSA Spread

• The most common form of spread is via the hands of healthcare workers.
• Precaution should be taken to perform rigorous hand washing.
• Elimination involves:
  • Administration of ointment mupirocin for nasal carriers.
  • Chlorhexidine washes for other sites of the body.

Treatment of MRSA

• Vancomycin.
• Daptomycin.

VISA

• Vancomycin-intermediate Staph. aureus.
• MIC for vancomycin is 4-8 μg/ml.
• Altered cell wall synthesis and structure.

VRSA

• Vancomycin-resistant Staph. aureus.
• MIC for vancomycin is ≥ 16 μg/ml.
• D-ala-D-ala conversion of D-ala-D-lactate/serine.
• VAN-A gene.

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Most Common Questions

Q: Is an indicator used in MSA?

Answer: C. Phenol red.

Q: Mannitol salt agar is a? 

Ans. A. Selective media.
B. Differential medium.

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