MICROBIOLOGY DIAGNOSTIC OF MICROORGANISMS RELATED TO CARDIAC INFECTIONS Microbiology Department

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

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

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

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

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

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

Blood sample BacilliCocciBacilliDiplococciOviod bodiesCocci Schema for the isolation and identification of the etiological agents of bacteremia Gram stain Observe for turbidity 3-to7-day trypticase soy broth culture, unvented Gram stain Observe for turbidity 3-to7-day trypticase soy broth culture, vented Brucella medium Brucella sp Blood agar (stab and streak inoculation) Hemolysis Streptococcus sp (for differentiation) Staphylococcus sp (for differentiation) MacConkey agar Enteric bacteria Lactosa fermention (-) (+) P.Aeruginosa Salmonella sp. E.coli H 2 S production (-) (+) P.aeruginosa Salmonella sp. Chocolate agar and CO 2 Oxidase test (see Exp.30) Neisseria sp. Sabouraud agar Lactophenol- cotton-blue stain (see Exp.36) C.albicans Blood agar Hemolysis Streptococcus sp. (for differentiation) Staphylococcus sp (for differentiation) (-)(+) (-) (+)

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

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

Staphylococcus

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

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

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

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

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

Tabel 1. Laboratory test for differentiation of Staphylococcal sp. TestS. aureusS.epidermidisS.saprophyticus MSA : -Growth -Fermentation Coagulase+-- DNase+-- Hemolysisbeta-- Novobiocin test SSR PigmentationGolden yellow white

Coagulase test

DNase Test

Blood agar

Bacteremic Pattern

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

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

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

Haemolytic activity : Alpha-haemolysis Beta-haemolysis Gamma-haemolysis

Haemolysis

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

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

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

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

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

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

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

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

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

Tabel 2. Laboratory Differentiation of Streptococci Group Organisms A S.ptogenes B S.agalactiae C S. equi D Enterococci E.faecalis Non- enterococci S.Bovis Hemolysisbeta Alpha- gamma Bacitracin test CAMP test-+--- Bile esculin hydrolysis % NaCl medium ---Growth- Growth at 10 o C ---Growth- Growth at 45 o C ---GrowthGrowth or -