MICROBIOLOGY DIAGNOSTIC OF MICROORGANISMS RELATED TO CARDIAC INFECTIONS

1. MICROBIOLOGY DIAGNOSTIC OF MICROORGANISMS RELATED TO CARDIAC INFECTIONS Microbiology Department

2. BLOOD CULTURE • Purpose • To become familiar with : • The microorganisms most frequently associated with bacteremia • Laboratory methods for the isolation and presumptive identification of the etiological agent of bacteremia

3. Collection of specimen : • Blood must be drawn aseptically • At least three blood sample from three different veni-puncture sites, separate from the last at least 1 hour • 10 ml blood should be collected from adult patient

4. PERICARDIAL FLUID • Purpose • To become familiar with : • Laboratory methods for the isolation and identification of the etiological agent of infective pericarditis

5. Collection of specimens : • Pericardial fluid must be collected aseptically • Should be injected immediately into : • Sterile tube or bottle • Anaerobic transport medium • Sterile heparin may be added to the fluid • Coagulated material should be emulsified

6. Microscopic examination : • Gram stain smear of the centrifuged sediment of clear slightly cloudy fluid should be examined • Purulent material should be smeared directly

7. Culture : The specimens should be streak into medium such as blood agar plate, Mc Conkey, chocolate agar, and Sabouraud agar plate, depend on the result of microscopic examination

8. Blood sample 3-to7-day trypticase soy broth culture, unvented 3-to7-day trypticase soy broth culture, vented Observe for turbidity Observe for turbidity Gram stain Gram stain (-) (-) (+) (+) Bacilli Cocci Bacilli Diplococci Oviod bodies Cocci Brucella medium Blood agar (stab and streak inoculation) MacConkey agar Chocolate agar and CO2 Sabouraud agar Blood agar Lactophenol- cotton-blue stain (see Exp.36) Brucella sp Hemolysis Enteric bacteria Hemolysis Oxidase test (see Exp.30) Streptococcus sp (for differentiation) Lactosa fermention (-) (+) Neisseria sp. C.albicans Streptococcus sp. (for differentiation) Staphylococcus sp (for differentiation) P.Aeruginosa Salmonella sp. E.coli Staphylococcus sp (for differentiation) H2S production (-) (+) P.aeruginosa Salmonella sp. Schema for the isolation and identification of the etiological agents of bacteremia

9. IDENTIFICATION OF HUMAN STAPHYLOCOCCAL PATHOGENS • Purpose • To become familiar with : • The medical significance of the staphylococci • Selected laboratory procedures designed to differentiate among the mayor staphylococcal species

10. Staphylococcus is : • Gram-positive cocci • Occur as irregular clusters • Non-spore-formers • Mesophilic bacteria • Resistant to drying

11. Staphylococcus

12. The three major species are : • S. aureus • S. saprophyticus • avirulent strain • S. epidermidis

13. Infection associated with S. aureus : • Skin infection : boils, carbuncles, acne, impetigo • Internal organ : pneumonia, cystitis, • tissue infection osteomyelitis, • pyelonephritis, • enteritis, septicemia, • endocarditis

14. Infection associated with : • S. epidermidis : skin lesions, • endocarditis • S. saprophyticus : urinary tract infection

15. S. Aureus metabolic end product : • Coagulase : • Bound coagulase (clumping factor) • Free coagulase • Leukocidin • Haemolysins • Enterotoxin

16. Non-toxic metabolites of S. aureus : • DNase • Lipase • Gelatinase • Staphylokinase

17. Tabel 1. Laboratory test for differentiation of Staphylococcal sp.

18. Coagulase test

19. DNase Test

20. Blood agar

21. Bacteremic Pattern

22. IDENTIFICATION OF HUMAN STREPTOCOCCAL PATHOGENS • Purpose • To become familiar with : • The medical significance of the streptococci • Selected laboratory procedures designed to differentiate streptococci on the basis of their hemolytic activity and biochemical patterns associated with the Lancefield group classifications

23. Streptococcus is : • Gram-positive cocci in chains • Nutritionally fastidious • Pinpoint colonies on solid media • Requiring enriched media for growth

24. The streptococci are classified • base on : • Their haemolytic activity • The serologic classification of Lancefield

25. Haemolytic activity : • Alpha-haemolysis • Beta-haemolysis • Gamma-haemolysis

26. Haemolysis

27. Alpha-haemolysis streptococci : • Incomplete form of haemolysis • Produce a green zone around the colony • Streptococcus viridans are non pathogenic opportunist • May produce sub-acute endocarditis • Streptococcus pneumoniae is the causative agent of pneumonia

28. Beta-haemolysis streptococci : • A complete destruction of red blood cells • Exhibit clear zone around the colony • Producing beta-haemolysins

29. Gamma-haemolytic • Gamma-haemolytic streptococci : • Absence of any haemolysis • Most commonly avirulent

30. Lancefield group classification : • Classified streptococci into 20 serogroups • Designated A through V (emitting I and J) • Base on the presence of C-substance, an antigenic group-specific hapten • Implicates the members of group A, B, C and D in human infectious processes

31. Group A : • Beta-haemolytic streptococci in this group referred to as streptococcus pyogenes • Main etiological agents of tonsillitis, bronchopneumonia, scarlet fever, erysipelas and cellulitis • Responsible for glomerulonephritis and rheumatic fever

32. Group B : • Beta-haemolytic streptococci indigenous to the vaginal mucosa • Responsible for puerperal fever, neonatal meningitis and endocarditis

33. Group C : • Beta-haemolytic streptococci • Have been implicated in erysipelas, puerperal fever, and throat infections

34. Group D : • Exhibit alpha or gamma-haemolysis • Includes enterococci such as Enterococcus faecalis • An etiological agent of urinary tract infections • The non-enterococci such as S. bovis

35. Extra-cellular metabolites of • streptococci : • Haemolysin (alpha and beta) • Leukocidins • Erythrogenic toxin • Hyaluronidase (spreading factor) • Streptokinase (a fibrinolysin) • Nucleases (ribonuclease and deoxybonuclease)

36. Tabel 2. Laboratory Differentiation of Streptococci

37. Thank You