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%
O A 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.

O To 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

O Ordering 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.
O After inoculating the culture vials, the vials are sent to the clinical pathology
microbiology department.
O Here the bottles are entered into a blood culture machine, which incubate the
specimens at body temperature.
O The 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 antibioticresistant strainsin particular,
methicillin-resistant S aureus (MRSA)
and, more recently, vancomycinresistant 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 methicillinresistant 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 firstgeneration 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, fibronectinbinding 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
O frusemide 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

O AND/OR
O spironolactone 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:

O enalapril 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
O Alpha/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
O Beta/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.
O Gamma - 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

Benzylpenicillin
Flucloxacillin
Penicillins

Cephalosporins

Glycopeptides

Narrow
spectrum

Bacterial meningitis
(Neisseria, streptococcus)
-Lactams
Contain a -lactam
ring; inhibit cell
wall synthesis by
binding to
transpeptidase,
inactivating it and
preventing crosslinking peptide
chains attached to
the peptidoglycan
backbone

Skin and soft tissue

infections (S. aureus, Strep)

Heart failure (high

sodium content)

Bronchitis, pneumonia, otitis

media, UTIs

Hypersensitivity
reactions

Ampicillin
Amoxycillin

Moderate
spectrum

Piperacillin
Ticarcillin

Broad
spectrum

Cephalexin

1st generation

Cefaclor

2nd generation

Ceftriaxone

3rd generation

Cefepime

4th generation

Pseudomonas aeruginosa

Structurally interferes with crosslinking of peptidoglycan backbone

Severe infections caused by

resistant bacteria, especially
MRSA; prophylactic in
endocarditis patients

Vancomycin

Serious infections with

Pseudomonas aeruginosa

Flucloxacillin is not
susceptible to -lactamase

Sinusitis
Septicaemia; pneumonia
Meningitis

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

Hypersensitivity
reactions

Ototoxicity,
nephrotoxicity

Augmentin = amoxicillin +
clavulanate clavulanate
competitively inhibits lactamase not susceptible
Alteration of the binding site so
that vancomycin cannot bind
(e.g. vancomycin-resistant
enterococcus, VRE)

Protein Synthesis Inhibitors

Tetracyclines

Tetracycline
Doxycycline

Aminoglycosides

Gentamycin
Tobramycin

Macrolides

Erythromycin
Roxithromycin
Clarithromycin

Bind and inhibit the function of the

30s ribosomal subunit,
preventing protein synthesis
Bind and inhibit the function of the
50s ribosomal subunit,
preventing protein synthesis

Rickettsial and chlamydial

infections

GIT disturbances,
Vit B deficiency

Listeria and P. aeruginosa

Ototoxicity,
nephrotoxicity
GIT disturbances,
hypersensitivity

Inactivation by microbial
enzymes, alterations of cell
membrane

Plasmid-controlled alteration of
the ribosomal binding site