Colloquium 1

• Staphylococci


  Live in the nasopharynx and skin of up to 50% of people
• Staphylococci


  • Family: Micrococcaceae
  • Genus: Staphylococcus
  • Many species, most important: S. aureus, S. epidermidis, S. saprophyticus
• Streptococci vs Staphylococci


  Need to differentiate so the correct Antibiotic can be prescribed
• 3 ways to differentiate Streptococci and Staphylococci

  1. Gram staining: Streptococci - Strip/Chain, Staphylocci - cluster

  2. Catalase: Streptococci Catalase negative, Staphylococci catalase positive

  3. Culture: S.aureus and certain Streptococci are B-hemolytic, S.aureus can be differentiated from other cocci due to golden pigment on sheep agar
• Coagulase test


  S.aureus is Coagulase positive (enzyme which activates prothrombin, causing a blood clot). This test allows you to differentiate gram positive cocci arranged in clusters. (coagulase negative indicates other Staphylocci eg epidermidis or saprophyticus)
• Glucose Broth


  • A. Staphylococcus spp. Culture (diffuse turbidity)
  • B. Non-inoculated broth (sterile)
• Thioglycate


  • A. Non-inoculated (sterile)
  • B. Inoculated with anaerobic bacteria (with bottom turbidity)
• Staphylococcus aureus


  • Smooth creamy (golden) colonies with beta-hemolysis
• Staphylococcus epidermidis

  • White, non-hemolytic colonies
• Plasma coagulase


  The major pathogenic factor of Staphylococcus aureus. When the isolate is cultured into rabbit plasma, after 2-6 hours at 37°C, released enzymes coagulate the plasma
• Positive coagulase test


  The tube is coagulated plasma following S. aureus inoculation
• Negative coagulase test


  When the tube is tilted with Staphylococcus spp. The plasma is liquid
• Pathogenetic Factors of Staphylococcus aureus

  • Proteins Disabling Immune Defences: Protein A, Coagulase, Hemolysins, Leukocidins, Penicillinase, Novel Penicillin binding proteins
  • Proteins - Tissue Damage: Hyalouronidase, Staphylokinase, Lipase, Protease
  • Exotoxin: Exfoliation, Enterotoxins, Toxic Shock Syndrome Toxin
• Diseases caused by Staphylococcus aureus

  • Diseases Caused by exotoxin release: Gastroentritis (food poisoning), Toxic Shock Syndrome, Skin Scalded Syndrome, toxic epidermal necrosis
  • Diseases resulting from direct organ invasion by the bacteria (non toxin mediated): Local or systemic, local pyogenic infections, Pneumonia, meningitis, osteomyelitis, Acute bacterial endocarditis, Septic arthritis, skin infections, bacteremia/sepsis, UTI
• Pathogenesis of Staphylococcus aureus infections

  • Transmission: via hands, aerosoles, blood, food
  • Entry: skin and mucosa, wounds, mouth, vagina, urethra
  • Dissemination - via blood in many organs and submucosa
• Microbiological diagnosis of S. aureus infections

  • Materials: pus-forming exudates, nasal swabs, blood culture, CSF, urine, feces, swabs from hospital environment, hands of medical personnel
  • Direct microscopy: only in CSF and primary sterile materials
  • Direct /fast/ tests for Ag detection: latex-agglutination and/or co-agglutination
  • Culture: Identification - morphological, cultural (golden pigmentation and beta-hemolysis), biochemical (catalase, coagulase). Automated systems.
  • Phage typing, plasmid typing or ribotyping (fingerprinting of genomic DNA restriction fragments) - in epidemics
• Staphylococcus epidermidis


  Part of normal bacterial flora, Coagulase negative, Normally lives peacefully on skin without causing disease. However hospital patients with Foley Urine catheters ot IV lines can become infected when microorganism migrates form skin to tubing. Causes pyogenic local infections (pus forming), endoplastitis (catheter associated infection), endocarditis ((infection of artificial valves), peritonitis (peritoneal dialysis infection)
• Staphylococcus saprophyticus


  Normal flora of skin, Coagulase negative, 2nd most leading cause of UTI in sexually active young women, Causes Cystitis
• Prophylaxis


  • Hand disinfection
  • Skin disinfection
  • Catheter hygiene
• Treatment for Staphylococcus aureus

  • Beta Lactams
  • Glycopeptides (eg. Vancomycin, teicoplanin)
  • Cephalosporins V (5th gen) (eg, ceftaroline, ceftobiprole)
  • Linozolide
• MRSA is resistant to methicillin (tend to develop in hospitals where broadspectrum Antibiotics are used)
• Streptococci


  Family Deinococcaceae, Genus Streptococcus, Significant human pathogens: S. pyogenes, S. agalactiae, S. pneumoniae
• Classification of Streptococci


  • According to hemolysis on BA: Alpha Hemolytic, Beta Hemolytic, Gamma Hemolytic
  • Serological (Antigenic characteristics of the C carbohydrate): Group A, B, D, Pneumococcus, Viridans group
  • Ecological: Pyogenic, oral, gut and anaerobic streptococci
• Pathogenetic Factors of Group A Beta-hemolytic Streptococci (GAS)


  • Components of Streptococcal Cell wall/associated with bacterial body: C carbohydrate, M protein (80 types), F protein
  • Enzymes: Halouronidase, DNA-ase
  • Hemolysins: Streptolysin O, Streptolysin S
  • Exotoxins: Erythrogenic toxin, Streptokinase
• Diseases caused by Group A Beta-hemolytic Streptococci (GAS)


  • By local invasion and/or exotoxin release: Streptococcal pharyngitis, Streptococcal skin infections, Necratising Fasciitis, Scarlett fever, Streptococcal Toxic Shock Syndrome
  • Delayed Antibody mediated diseases (Post streptococcal complications): Rheumatic fever, Acute Glomerulonephritis
• Group B Streptococci (eg; S.agalacticae)

  Also B-hemolytic, 25% of women carry these bugs vaginally, baby acquire it during delivery. Causes neonatal meningitis, pneumonia, sepsis
• Viridans Group Streptococci


  No Lancefield Ag classification, Normally human GIT flora, Causes dental infection, endocarditis, abscesses
• Microbiological diagnosis of streptococcal infections

  • Materials: nasopharyngeal swabs, CSF, blood culture, urine, genital swabs, pus, wounds
  • Direct microscopy - in primary sterile materials
  • Culture - BA, glucose broth
  • Identification: catalase /-/
  • For group A and B: presumptive - morphological, cultural - hemolysis, bacitracin sensitivity, confirmative - hippurate hydrolysis, CAMP test
  • For viridans streptococci: differentiation from pneumococci - optochin test, bile sensitivity, inulin, inoculation in mouse
  • For enterococci: esculine hydrolysis, growth in milk with methylene blue, 6.5% NaCl, 9.6 pH
  • Serology: antistreptolysin titer in rheumatism (AST)
• CAMP test


  An enhanced zone of beta-hemolysis of Staphylococcus spp. Is observed when cultured with S. agalactiae. Used for identification of Streptococcus beta-hemolyticus group B (Streptococcus agalactiae)
• Prophylaxis


  • Hand disinfection, non specific immune stimulators
• Treatments


  • GAS: Penicillin (10-day treatment strep pharyngitis)
  • PRSP: Cephalosporins
  • Macrolides are also used (eg, azithromycin, Clarithromycin)
• Streptococcus pneumoniae


  No Lancefield Ag, Morphology - ovoid or lancet-shaped cocci in pairs, Cultures - colonies with alpha-hemolysis, transparent with autolysis, Biochemical activity - inulin fermentation, catalase negative, Resistance - low, Sensitive to bile
• Esculine hydrolysis


  Growth in milk with methylene blue, 6.5% NaCl, 9.6 pH
• Antistreptolysin titer


  Test used in rheumatism
• CAMP factor


  Increases haemolysis in red cells pre-treated with S.aureus toxin
• CAMP factor test is used for identification of Streptococcus beta-hemolyticus group B (Streptococcus agalactiae)
• An enhanced zone of beta-hemolysis of Staphylococcus spp. is observed when cultured with S. agalactiae
• Prophylaxis


  • Hand disinfection
  • Non specific immune stimulators
• Treatments


  • Penicillin (10-day treatment strep pharyngitis)
  • Cephalosporins for PRSP
  • Macrolides (eg, azithromycin, Clarithromycin)
• Streptococcus Pneumoniae


  No Lancefield Ag