Blood%culturing%&%microbiology%

Blood%culturing%OBlood culture is a microbiological culture of blood.

It is employed to detect infections that are spreading through the bloodstream (such as bacteremia, septicemia amongst others)

OThis is possible because the bloodstream is usually a sterile environment

OWhen a patient shows signs or symptoms of a systemic infection, results from a blood culture can verify that an infection is present, and they can identify the type (or types) of microorganism that is responsible for the infection

Method%of%blood%culturing%OA minimum of 10 ml of blood is taken through venipuncture and injected into two or

more "blood bottles" with specific media for aerobic and anaerobic organisms. O A common medium used for anaerobes is thioglycollate broth.

OTo maximise the diagnostic yield of blood cultures, multiple sets of cultures (each set consisting of aerobic and anaerobic vials filled with 310 mL) may be ordered by medical staff.

O A common protocol used in US hospitals includes the following: O Set 1 = left antecubital fossa at 0 minutes O Set 2 = right antecubital fossa at 30 minutes O Set 3 = left or right antecubital fossa at 90 minutes

OOrdering multiple sets of cultures increases the probability of discovering a pathogenic organism in the blood and reduces the probability of having a positive culture due to skin contaminants.

OAfter inoculating the culture vials, the vials are sent to the clinical pathology microbiology department.

OHere the bottles are entered into a blood culture machine, which incubate the specimens at body temperature.

OThe blood culture instrument reports positive blood cultures (cultures with bacteria present, thus indicating the patient is "bacteremic"). Most cultures are monitored for five days, after which negative vials are removed.

If%positive%OIf a vial is positive, a microbiologist will perform a Gram

stain on the blood for a rapid, general identification of the bacteria, which the microbiologist will report to the attending physician of the bacteremic patient.

O The blood is also subcultured or "subbed" onto agar plates to isolate the pathogenic organism for culture and susceptibility testing, which takes up to three days.

O This culture and sensitivity (C&S) process identifies the species of bacteria. Antibiotic sensitivities are then assessed on the bacterial isolate to inform clinicians with respect to appropriate antibiotics for treatment.

Infective%endocarditis%and%Microbiology%

OBacteraemia leads to colonisation of the thrombus and perpetuates further fibrin deposition and platelet aggregation, which develops into a mature infected vegetation.

OAcute IE is usually associated with more virulent organisms, classically Staphylococcus aureus. Thrombus is formed by the offending organism and S aureus may invade endothelial cells and increase the expression of adhesion molecules as well as prothrombotic factors

S.%aureus%OA gram-positive coccal bacterium OResponsible for many infections but it

may also occur as a commensal. OBoth community-associated and

hospital-acquired infections with Staphylococcus aureus have increased in the past 20 years, and the rise in incidence has been accompanied by a rise in antibiotic-resistant strainsin particular, methicillin-resistant S aureus (MRSA) and, more recently, vancomycin-resistant strains

O Can affect many different parts of the body

Resistant%strains%OMethicillin-resistant S. aureus [MRSA] is one of a number

of greatly feared strains of S. aureus which have become resistant to most -lactam antibiotics.

OMRSA strains are most often found associated with institutions such as hospitals, but are becoming increasingly prevalent in community-acquired infections

OFor this reason, vancomycin, a glycopeptide antibiotic, is commonly used to combat MRSA. Vancomycin inhibits the synthesis of peptidoglycan, but unlike -lactam antibiotics, glycopeptide antibiotics target and bind to amino acids in the cell wall, preventing peptidoglycan cross-linkages from forming.

OVancomycin-resistant S. aureus (VRSA) is a strain of S. aureus that has become resistant to the glycopeptides.

S.%Aureus%&%Infective%endocarditis%

OSigns & symptoms of endocarditis ! Initially presents as fever and malaise; peripheral emboli may be present; may involve healthy valves

ODiagnosis of S. aureus ! Blood culture is the most important diagnostic procedure OInject the blood sample into hypertonic media if the

patient has been exposed to antibiotics OObtain 3-5 sets of large-volume blood cultures within the

first 24 hours OEchocardiography is a valuable adjunct

Management%of%S.%aureus%OEmpiric therapy with penicillins or cephalosporins may be

inadequate because of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) OCombination therapy with a penicillinase-resistant penicillin or

cephalosporin (in case the organism is methicillin-sensitive S aureus [MSSA])

OFor Endocarditis OThe combination of a beta-lactam and an aminoglycoside (eg, nafcillin

and gentamicin) OIn patients with MRSA, combinations of vancomycin with

aminoglycosides ORifampin can be added to combination therapy, especially for

prosthetic valve endocarditis ODuration of therapy is at least 4 weeks OBacteremia, fever, and leukocytosis for at least a week after therapy is

initiated

Treatment%continued%OSerious staphylococcal infections require

treatment with parenteral penicillinase-resistant penicillin (eg, nafcillin, oxacillin) or first-generation or second-generation cephalosporins (eg, cephalexin, cefuroxime) plus clindamycin.

OVancomycin is reserved for staphylococcal strains that are resistant to penicillinase-resistant penicillins (ie, MRSA) and clindamycin, or for when the patient has potentially life-threatening infection or intoxication

Strep.%pyogenes%OStreptococci are a large group of gram-positive, nonmotile,

nonspore-forming cocci about 0.5-1.2m in size. They often grow in pairs or chains and are negative for oxidase and catalase.

Otends to colonize the upper respiratory tract and is highly virulent as it overcomes the host defense system. The most common forms of S pyogenes disease include respiratory and skin infections, with different strains usually responsible for each form

OBacterial virulence factors: The cell wall antigens include capsular polysaccharide (C-substance), peptidoglycan and lipoteichoic acid (LTA), R and T proteins, and various surface proteins, including M protein, fimbrial proteins, fibronectin-binding proteins (eg, protein F), and cell-bound streptokinase

Rheumatic%Heart%Disease%&%Streptococcus%pyogenes%

ORheumatic fever is a late inflammatory, nonsuppurative complication of pharyngitis that is caused by group A-hemolytic streptococci.

ORheumatic heart disease is cardiac inflammation and scarring triggered by an autoimmune reaction to infection with group A streptococci OStreptococcal proteins display molecular mimicry recognized by the immune

system, especially bacterial M-proteins and human cardiac antigens such as myosin and valvular endothelium. Antimyosin antibody recognizes laminin, an extracellular matrix alpha-helix coiled protein, which is part of the valve basement membrane structure.

OT-cells that are responsive to the streptococcal M-protein infiltrate the valve through the valvular endothelium, activated by the binding of antistreptococcal carbohydrates with release or tumor necrosis factor (TNF) and interleukin

Management%of%RHD%OFor carditis, the most important initial aspect is management of any

cardiac failure. A combination of bed rest, fluid restriction and diuretics is appropriate for mild to moderate heart failure

OCheck that renal function is normal then use Ofrusemide 1 to 2 mg/kg orally as a single dose, then 0.5 to 1 mg/kg (to a

maximum of 6 mg/kg) orally, 6- to 24-hourly OAND/OR Ospironolactone 1 to 3 mg/kg (initially) up to 200 mg orally, daily in 1 to 3

divided doses. Round dose to a multiple of 6.25 mg (a quarter of a 25 mg tablet).

O Continue until the cardiac failure is controlled and the carditis improved. OFor more severe cardiac failure, an angiotensin converting enzyme

(ACE) inhibitor should be added. For example: Oenalapril 0.1 mg/kg (adult: 2.5 mg) orally, daily in 1 or 2 divided doses

increased gradually over 2 weeks to a maximum of 1 mg/kg (adult: 10 to 20 mg) orally, daily in 1 or 2 divided doses

O lisinopril 0.1 to 0.2 mg/kg (adult: 2.5 to 20 mg) orally, daily up to a maximum of

1 mg/kg (adult: 40 mg) orally, daily.

Classication%of%Streptococcus%species%Haemolysis - The ability of bacterial colonies to induce hemolysis when grown on blood agar is used to classify certain microorganisms OAlpha/partial haemolysis - the agar under the colony is dark and greenish. Streptococcus

pneumoniae and a group of oral streptococci display alpha hemolysis. caused by hydrogen peroxide produced by the bacterium, oxidizing hemoglobin to green methemoglobin

OBeta/complete haemolysis a complete lysis of red cells in the media around and under the colonies: the area appears lightened (yellow) and transparent. O Streptolysin, an exotoxin, is the enzyme produced by the bacteria which causes the

complete lysis of red blood cells. There are two types of streptolysin: Streptolysin O (SLO) and streptolysin S (SLS). OStreptolysin O is an oxygen-sensitive cytotoxin, secreted by most Group A streptococcus (GAS),

and interacts with cholesterol in the membrane of eukaryotic cells (mainly red and white blood cells, macrophages, and platelets), and usually results in -hemolysis under the surface of blood agar.

OStreptolysin S is an oxygen-stable cytotoxin also produced by most GAS strains which results in clearing on the surface of blood agar. SLS affects immune cells, including polymorphonuclear leukocytes and lymphocytes, and is thought to prevent the host immune system from clearing infection. Streptococcus pyogenes, or Group A beta-hemolytic Strep (GAS), displays beta hemolysis.

OGamma - does not induce hemolysis, the agar under and around the colony is unchanged, and the organism is called non-hemolytic or said to display gamma hemolysis (-hemolysis) e.g. Enterococcus faecalis

Pharmacology of Antibiotics Antibiotics Type/Mechanism Used On Unwanted effects Resistance

Cell Wall Inhibitors

Penicillins

Benzylpenicillin Narrow spectrum -Lactams

Contain a -lactam ring; inhibit cell wall synthesis by binding to transpeptidase, inactivating it and preventing cross-linking peptide chains attached to the peptidoglycan backbone

Bacterial meningitis (Neisseria, streptococcus)

Heart failure (high sodium content)

Hypersensitivity reactions

1. Destruction by microbial -lactamase

2. Failure to reach the target transpeptidase alteration of porins to reduce entry

3. Failure to bind to the transpeptidase poor affinity

Flucloxacillin is not susceptible to -lactamase Augmentin = amoxicillin + clavulanate clavulanate competitively inhibits -lactamase not susceptible

Flucloxacillin Skin and soft tissue infections (S. aureus, Strep)

Ampicillin Amoxycillin

Moderate spectrum

Bronchitis, pneumonia, otitis media, UTIs

Piperacillin Ticarcillin

Broad spectrum

Serious infections with Pseudomonas aeruginosa

Cephalosporins

Cephalexin 1st generation Sinusitis

Hypersensitivity reactions

Cefaclor 2nd generation Septicaemia; pneumonia

Ceftriaxone 3rd generation Meningitis

Cefepime 4th generation Pseudomonas aeruginosa

Glycopeptides Vancomycin Structurally interferes with cross-linking of peptidoglycan backbone

Severe infections caused by resistant bacteria, especially MRSA; prophylactic in endocarditis patients

Ototoxicity, nephrotoxicity

Alteration of the binding site so that vancomycin cannot bind (e.g. vancomycin-resistant enterococcus, VRE)

Protein Synthesis Inhibitors

Tetracyclines Tetracycline Doxycycline Bind and inhibit the function of the 30s ribosomal subunit, preventing protein synthesis

Rickettsial and chlamydial infections

GIT disturbances, Vit B deficiency Inactivation by microbial

enzymes, alterations of cell membrane Aminoglycosides Gentamycin Tobramycin Listeria and P. aeruginosa

Ototoxicity, nephrotoxicity

Macrolides Erythromycin Roxithromycin Clarithromycin

Bind and inhibit the function of the 50s ribosomal subunit, preventing protein synthesis

GIT disturbances, hypersensitivity Plasmid-controlled alteration of the ribosomal binding site