MICROBIAL INFECTION IN

CARDIOVASCULAR SYSTEM
FADHILAH
FACULTY OF MEDICINE, HASANUDDIN UNIVERSITY
NOVEMBER 2021
 Aims

After presenting this lecture, the students must

be able to explain about the characteristics,
virulence factors, pathogenesis of microbial
infection causing cardiovascular disorder,
clinical manifestation, laboratoric diagnosis
and the treatment for cardiovascular infectious
diseases.
Infections of the cardiovascular system:
• Myocarditis
• Pericarditis
• Endocarditis
INFECTIOUS MYOCARDITIS
 Myocarditis
• Myocarditis is an inflammation of the
myocardium, may be caused by:
– infectious agents (bacterial, mycotic, viral,
parasitic)
– noninfectious: unknown/idiopathic, toxin/drug
reaction, autoimmune
Myocarditis
Acute : symptoms ranges from an asymptomatic illness
with reversible changes to fulminant myocardial
necrosis and death
Chronic : lymphocytic infiltration of the myocardium
may cause subacute/chronic deterioration of cardiac
function
 Microorganisms causing Myocarditis
• Virus: Parvovirus B19, HHV6, Coxsackie
Virus
• Fungi
• Bacteria
– Bacterial toxins (diphteria toxin)
– The Spirochetes (Borrelia burgdorferi causing tick
born Borrelia Lyme myocarditis)
– Rickettsiae (Rickettsia ricketsii)
• Parasites (toxoplasmosis, Trypanosoma Cruzi)
 Infectious causes of Myocarditis
Region Normal host Immunocompromised
Developed VIRUSES: Coxsackie A and B, echovirus, VIRUSES: HIV, CMV, EBV,
World Common CMV, EBV, HHV-6, Influenza virus A and VZV, adenovirus,
B, adenovirus, Parvovirus, HBV, HCV parvovirus
BACTERIA: diphteria, Lyme disease, FUNGI: Candidiasis,
organisms associated with IE aspergillosis,
PARASITES: American Trypanosomiasis, cryptococcosis
trichinosis PARASITES:toxoplasmosis,
American trypanosomiasis

Unco
mmon
VIRUSES: Adenovirus, Parvovirus, FUNGI:
Respiratory syncitial virus, HBV, HCV histoplasmosis,
BACTERIA: Staphylococci, Streptococci, blastomycosis,
meningococci, Salmonellae, listeria, coccidioidomycosis,
clostridia, rickettsia, bartonellosis, zygomycosis
ehrlichiosis

Developing VIRUSES: poliovirus, mumps, rubella, arenaviruses, dengue, rabies,

World chikungunya, ebola virus, yellow fever
BACTERIA: leptospirosis
PARASITES: American Tryp,. African Trypanosomiasis
 Viral cause of Myocarditis
• Viral myocarditis:
– Usually mild or asymptomatic
– Cardiac Signs: due damage to the myocard
(cardiomegaly, short breath, palpitations, arythmia)
– Non-cardiac signs: rash, fever, sore throat
– Chest pain
PERICARDITIS
 Pericarditis
• Pericarditis: injury to the pericardium causing cellular
infiltration, fibrin deposition and outpouring of pericard
fluid.
• The cause of pericarditis:
– Infectious agents
– Non-infectious: Acute MI, Uremia, neoplasm, myxedema,
cholesterol, trauma, aortic aneurism (with leakage to
peric.sac), radiation, assoc with severe chronic anemia,
infectious mononucleosis, sarcoidosis, postcardiac surgery,
acute idiopathic.
– Hypersensitivity /autoimmunity: RF, collagen vascular
disease (SLE, RA, scleroderma), drug-induced (procainamid,
hidralazin), Post MI (Dressler’s syndrome,
postpericardiotomy)
Infectious Agents causing Pericarditis
– VIRAL
– BACTERIAL: pyogenic or syphilitic
– TUBERCULOUS
– MYCOTIC
– PARASITIC
 Infectious causes of pericarditis
Viruses (most common cause of idiopathic Mycobacteria
Pericarditis)
CMV, Mycobacterium tuberculosis,
Herpes simplex virus, Mycobacterium chelonae, Mycobacterium
Coxsackie A, B, avium complex
Echovirus,
Epstein Barr virus, Spirochetes
adenovirus,
Influenza,
Mumps, VZV, EBV, and HIV
Borrelia burgdorferi

Bacteria Mycoplasma

Streptococcus pneumoniae, Mycoplasma pneumoniae, Ureaplasma

Streptococcus spp., Staphylococcus urealyticum, Mycoplasma hominis
aureus, Neisseria meningitidis, Fungi
Listeria monocytogenes, Hemophilus Histoplasma capsulatum, Coccidioides
influenzae, Francisella tularensis, immitis, Cryptococcus neoformans,
Brucella melitensis, Enteric Gram Blastomyces dermatitidis, Candida spp.,
negative rods, Actinomyces spp., Aspergillus fumigatus
Nocardia asteroides, legionella Parasites
pneumophila, Tropheryma whippelii,
Toxoplasma gondii, Entamoeba histolityca,
Salmonella spp., Campylobacter spp., Echinococcus granulosus, Schistosoma spp
Rickettsia/Q fever
 Bacterial causative of pericarditis
• Bacteria spread to the pericard through:
– Hematogenous seeding during bacteremia (esp Mtb)
– Extension of infection from a contiguous focus in the chest
(complication postoperative or post-traumatic) or from a
subdiaphragmatic abscesses
– Invasive bacterial infection (staphylococcal endocarditis)

Pathologic changes in the pericardium which are caused by

immune complex deposition  sterile exudates
Most effusions resolve without specific therapy
Most common bacteria: streptococci (S.pnie, S. Vir.)** and
staphylococci
 Causes of pericarditis:

• Mycoplasma causing pericarditis (Fournier et al, 2001) Treat

with doxycyclin after drainage
• Mycobacteria: important cause in developing countries; 1-8% cases
among pulmonary tuberculosis patients.
• Histoplasma capsulatum; 6% of symptomatic histoplasmosis
Laboratory Findings and Diagnostic Studies

• VIRAL PERICARDITIS:
– Clinical
– Leukocytosis
– Rising titer in paired sera  rarely done
• TUBERCULOUS PERICARDITIS: Inferred
diagnosis if AFB is found elsewhere.
• BACTERIAL PERICARDITIS:
pericardiocentesis
INFECTIVE ENDOCARDITIS
 Infective Endocarditis
Inflammation or infection of the endocard including the valve
And Nonvalvular areas or implanted mechanical devices

caused by microorganisms

-Artific.Heart valve
-Pacemakers
-Implantable defibrillators
preexisting tissue damage

frequently fatal
 KEY POINTS

• Infective endocarditis (IE) remains universally

lethal if not aggressively treated
• Medical progress has altered the epidemiology
of IE
• Successful therapy for IE is being challenged
by the development of antibiotic resistance
 Epidemiology of IE
• Relatively rare – due to lacking report?
• In developing countries - before year 1950 - IE
was a complication of Rheumatic Heart Disease
and poor dentition.
• Antibiotic use  demoraphic pattern changed from 30-40
in the preantibiotic era to 47-69 in the 1st decade of the
21st century.
• Aging  more Degeneration Heart Valve disease  more
use of heart planted substitutes and intracardiac devices.
• Predisposing chronic comorbidities (diabetes, HIV, renal
disease, nosocomial bacteremia).
• IE is IMPORTANT because of its serious
complications:
– Stroke
– Requires open heart surgery
– Death
 Pathogenesis of IE
• Persistent endocardial infection 
continous bacteremia
• Is uncommon due to transient bacteremia.
 Endocarditis
• Based on clinical symptoms
• Acute Bacterial Endocarditis – acute and fulminant
– Caused by Staphylococcus aureus (>80% cases)
– Virulent  May even affect healthy valves
• Subacute Bacterial Endocarditis
– Caused by Streptococcus viridans, and HACEK (1.2 – 3%
cases)
– Clinical symptoms unnoticable/more Insidious
– Usually affect already damaged valves

Recently, due to lack of clinical importance to distinguish acute and sub acute,
now this classification is not further discussed. ,
• Based on the host
– Native valve endocarditis (NVE):
•  Streptococcus viridans, Group D streptococcus,
S.aureus, Enterococci and HACEK, or rarely gram
negatives eg. Eschericia coli
– Prosthetic valve endocarditis (PVE)
Staphylococcus epidermidis
– Drug Addicted persons endocarditis (IVDA) 
usually Staphylococcus aureus or fungi
• Echocardiography helps to
visualize the heart valves and
deformities.
• Vegetations growing on the
valve may damage the function
of the valve.

Excised valve

Ref: Cabell , et al, 2003

 How IE Occurs
Injured endocard surface

Sterile thrombus develops on the injured surface =vegetation

= nonbacterial thrombotic endocarditis= NBTE

Bacterial entrance to the blood

(after brushing teeth/injury/diagnostic procedure)
certain bacteria attaches to the injured surface

Fibrin and platelets deposited

Complications:
Mechanical cardiac injury, thrombotic/septic emboli, immune injury
 Lesions on the heart that predispose Endocarditis

1. Rheumatic Valvular disease

2. Acquired valve lesions
3. Hypertrophic obstructive cardiomyopathy
4. Congenital Heart disease (PDA, VSD, TF, Bicuspid aortic
valves)
5. Surgically implanted intravascular hardware, prosthetic heart
valves, pulmonary systemic vascular shunts, ventriculo-atrial
shunts for hydrocephalus
6. Previous endocarditis
Factors that enables any microorganism
causing IE:

• 1. Ability to enters the circulation

• 2. Ability to survive in the blood
• 3. Ability to attach to the endocard
Microorganisms causing IE:

Ref: Sharara et al, 2016

Most common causing microorganisms
are GRAM positives (90%):
:
– Staphylococcus aureus & Coagulase neg
staphylococcus)
– Streptococcus spp. Why?
– Enterococci
• Because of its resistance to elimination and because of its dextran
component of the cell wall that can be used for attachment to
thrombus
• Less common cause (10%):
– Coxiella burnetii (Q fever)
– Chlamydia spp
– Legionella spp
– Bartonella spp
– HACEK
– fungi
 How do Streptococci avoid clearance?
Blood flow can clear bacteria within minutes and detoxify
bacteria

Released adhesins allow adherence

to platelet’s membrane

Secreted Platelet Aggregation

Associated Proteins induces platelet
aggregation especially on injured
heart valve
Released ADP and serotonin allows
recruitment of additional platelets streptococci trapped
within
the thrombus.
Upregulation of aII b63-integrin
binds fibrinogen, forming a thrombus .
 Subacute IE
Caused by gram negative, nasal/urogenital normal flora
(HACEK)
Symptoms occurs from a few days to 13 weeks between the
identifiable event producing bacteremia to the time of
diagnosis
Risk factors: preexisting cardiac disease (60%), prosthetic valve,
native valve (80%), history of Rh.fever, dental procedures.
Low grade fever
Larger vegetation finding because later time of D/
Better prognosis than non HACEK pathogens
without other co morbidities
Younger age
Community acquired
Microbiological Methods for diagnosis of
infection

• Bacterial detection:
– Culture, isolation and identification of specimen
– Serologic examination of serum (Antibody titer)
– Molecular  Polymerase Chain Reaction (PCR)
• Viral detection
– Tissue culture
– Serologic examination of serum
– Molecular (PCR)
• Fungal
– Culture, isolation and identification
– Molecular (PCR)
 Blood Culture
• Requires +10 ml blood, collected 3 times within 24 hours and at
least 1 hour apart; before antibiotics
• If clinically stable, stop antibiotics 2-3 days before collecting blood
for culture
• Causes of negative culture:
 Fungus
 previously treated with antibiotic
 caused by fastidious m.o. (Legionella, Bartonella, abiotrophia)
 Bacteria can not grow in artificial media
 Slow grower bacteria  2 -21 days for HACEK (6 days of blood culture)
• If negative Culture  should order:
– Serology
– tissue culture (to find intracellular bacteria Coxiella burnetii, Chlamydia
spp., legionella spp., Bartonella spp..
– immunohistology
– PCR detection
• Tissue culture
 Serology tests
• Agglutination test for Brucella melitensis
• Indirect fluorescense for L. pneumophila
• ELISA for Mycoplasma pneumoniae
• CF, ELISA and indirect IF for Chlamydia spp..
 Management of IE:

– Antibiotics appropriate to the causing bacteria:

Amox/Ampicillin + aminoglikosida or
Cephalosporins 3rd generation- 4 w for native
valve, 6 w for prosthetic valve
– Surgery to remove infected tissues or correct the
damaged valve; the indications are:
• Refractory cardiac failure
• Persistent sepsis caused by a surgically removable
focus or a valvular ring or myocardial abscess
• Persistent life-threatening embolization
Clinical course of Endocarditis
Clinical Course of endocarditis
Clinical Course of endocarditis
Acute Rheumatic Fever and Rheumatic Heart
Disease
• Inflammatory disease affecting the heart, skin and
soft tissues
• Commonly attacks children or young adults 
peak incidence at 5-15 y.o.
• As a complication (occurring 5 days-10 w;
usually 2-3 w) after pharyngitis which is caused
by Group A Streptococcus pyogenes
• Diagnosis: Jones Criteria and confirmation of
streptococcal infection
 Pathogenesis of ARF/RHD

• Toxins secreted by Streptococci

• Autoimmune cross reactions between bacterial antigens
and endocard

• Inflammation may affect pericard, myocard and

endocard Scarred tissue (75-80% attacks the mitral
valve) deformity
• Defect on valves do not appear until 10-30 years (in
developed country latency period is shorter)
• Recurrent in 10%  more damage to the heart 
Prophylaxis is important
 sudden onset of sore throat Strep infection Asymptomatic
pain on swallowing with symptoms strep infection
fever
headache streptokinase
red throat/tonsils streptolysin O (SLO)
abdominal pain, nausea and Infectious
DNAase
vomiting, especially in children Infection of the pharynx materials of
Group A Hyaluronidase
streptococci major surface protein,
M protein

Infection subsided
Ab to strept +

?
Antibiotics Persistent
are required eventhough Strep in the throat
symptoms disappeared
Hyaluronic acid
Ongoing A b producing
capsule
esp. anti M Ab
fever
painful, tender, red swollen joints Causing autoreactivity
pain in one joint that migrates to another one
heart palpitations Acute rheumatic fever
chest pain 
shortness of breath
skin rashes
fatigue
small, painless nodules under the skin  Rheumatic Heart Disease &
Post streptococcal
Rheumatoid arthritis
REVIEW ON MICROBIAL AGENTS OF
CARDIOVASCULAR INFECTIONS
Diseases caused by streptococci (1)
• Acute Streptococcus pyogenes infections:
– pharyngitis, scarlet fever (rash), impetigo,
cellulitis, or erysipelas.
– Invasive infections necrotizing fasciitis, myositis
and streptococcal toxic shock syndrome.
– Immune-mediated sequelae  acute rheumatic
fever and acute glomerulonephritis. 
Disease caused by streptococci (2)
• Streptococcuc agalactiae :
– Neonates: meningitis, neonatal sepsis, and
pneumonia in neonates
– Adults: vaginitis, puerperal fever, urinary tract
infection, skin infection, and endocarditis.
Viridans group streptococci: endocarditis
Enterococcus: urinary tract and biliary tract infections.
Anaerobic streptococci : mixed infections of the
abdomen, pelvis, brain, and lungs.
 Streptococcus
• Family: Streptococcaceae
• Genus: Streptococcus Small colonies appearance on Blood agar:
This shows gamma hemolytic streptococci

• Morphology:
– Coccus, gram positive
– On solid media: circular colony, convex, translucent-
opaque, pinpoint size (0.5-1 μm)
– Catalase test negative
– Requires enriched medium: blood agar 5%
– Facultative anaerobe, some are strict anaerobe
– In broth medium, they grow in pairs or chains
 Pathogenicity of streptococci
• Virulence factors of the cell:
– Adherence factors
– Toxins (exotoxin)
– Enzymes
– Antiphagocytic factors
 Cell structure of Streptococci

Electron
microscopy of
streptococci,
 Antigenic structures of the cell
• Capsule: hyaluronic acid polysacharide
• mask its antigenicity from phagocytes  no phagocytosis
• Binds to CD44 of the respiratoric epithelial cells
• Cell wall: peptidoglycan
• Lipotheichoic acid (LTA)
• Adherent factor, for invasion, inhibit phagocytosis
• Protein M – hairlike outward protrusion from inside the cell wall
• inhibits opsonization of the complement system 
inhibits phagocytosis
• Protein F – fibronectin binding protein  respiratory
cell attachment
• Extracellular substances: exotoxins and enzymes
Exotoxins and enzymes produced by
Streptococcus
1. Haemolysin -- alpha and beta
2. Leucocydin – destroys phagocytes
3. Erythrogenic/pyrogenic toxin  scarlet
fever
4. Hyaluronidase – hydrolyse tissue
cement/hyaluronic acid
5. Streptokinase – fibrinolysin
6. Nuclease (ribonuclease,
dioxyribonuclease) – destroys viscous
tissue debris
 Classification of streptococci
Classf is based on:
• Colony morphology
• Haemolysis
• Biochemical reactions
• Serologic specificity  most definitively .. based
on antigenic differences in cell wall carbohydrates
(groups A to V), in cell wall pili-associated
protein, and in the polysaccharide capsule in
group B streptococci.
 Classification based on hemolysins
produced
• Hemolytic activity:
– α hemolytic
– Β hemolytic
– γ hemolitic
 Αlpha hemolytic
• incomplete hemolysis; green zone around
colony; oxydation of iron in hemoglobin
(Hb methHb)
e.g. Viridans Group of streptococci, usually
nonpathogenic but opportunistic
 Βeta hemolytic
o Complete hemolysis of blood, clear zone around
colonies, 2-4 x larger the size of colony.
o Streptococcus pyogenes (member of GAS = Group A
streptococcus) – causing tonsillitis,
bronchopneumonie, scarlet fever, erysipelas, cellulitis,
glomerulonephritis, rheumatic fever
o Streptococcus betahemolytic among GBS are those
found in vaginal mucosa causing puerperal infection,
neonatal meningitis, endocarditis
o Streptococcus betahemolytic among group C are those
causing erysipelas, puerperal fever, throat infections
Streptococcus (Cat -, Coag - ) grouping based on cell
wall’s carbohydrate antigenic differences (A-V)

Group A - Streptococcus pyogenes

Group B - Streptococcus agalactiae
Group C - Streptococcus equisimilis, Streptococcus equi,
Streptococcus zooepidemicus, Streptococcus dysgalactiae
Group D -Enterococcus faecalis, Enterococcus faecium,
Enterococcus durans and Streptococcus bovis
Group E - Enterococci
Group F, G & L - Streptococcus anginosus
Group H - Streptococcus sanguis
Group K - Streptococcus salivarius
Group L - Streptococcus dysgalactiae
Group M & O - Streptococcus mitior
Group N - Lactococcus lactis
Group R & S - Streptococcus suis
 PYR neg
PYR pos PYR pos
Distinguishing S.pyogenes and Enterococcus
sp. from S.agalactiae

PYR test = Pyrrolidonil arylamidase test  Presumptive Rapid test

 10 mins in strips; in broth 4 hrs
1. Add a loop of isolated colony to broth
L naphtylamide B naphtylamide in broth is
hydrolysed by streptococci’ aminopeptidase 
releasing free naphtylamide
2. Add a drop of NN dimethyl aminocinnamaldehyde 
cherry colour  S.pyogenes
 Distinguishing S. pneumoniae from
Streptococci group viridans
• Optochin Test
– Optochin lyses Streptococcus pneumoniae
– Streak streptococci suspension on MHA (mueller hinton agar)
– Place Optochin disc on agar, incubate 24 hours
observe zone of growth inhibition (must be >14 mm)

• Bile Solubility Test

– Differentiate S. pneumoniae from other alphahemolytic strains (viridans) 
see also BA* plate
– Bile or Sodium desoxycholate (bile salt) reduces surface tension  cell
lysis. Bacterial autolytic enzymes results in a faster lytic action because of
the lowered bacterial & medium’s surface tension
• Grow bacteria 13-24 hours (not longer or colonies will grow old) on Blood Agar
• Place a drop of 10% bile salt next to an isolated colony
• Gently allow liquid cover the colony, do not dislodge colony
• Incubate at 35oC, aerob, for 30 minutes -- Observe lysis of colony
• Test tube method: prepare 0.5 MacFarland bacterial suspension  in one tube 0.5 ml
add with 0.5 ml 2% bille salt, in another tube 0.5 ml add with 0.85% NaCl  incubate
35oC 10 minutes or upto 2 hours  see clearing of cells  Pos
Differentiating Streptococcus pneumoniae
from Streptococcus of the viridans group

• S.pneumoniae after 24-48 hours produce

indented surface; S.viridans’ surface
remains domed.
 Bile esculin test
• Pos test: Group D streptococci (S.bovis) and
enterococcus
– Bacteria able to hydrolize esculin even in the
presence of bile  bacteria grows  forms
glucose and esculetin  Esculetin reacts with
ferric ions  media turns black
• Neg test: Non Group D streptococci (viridans
group) cannot hydrolize esculin
Distinguishing S. pneumoniae from
Streptococci of the viridans group
 Group A Streptococcus
• Protein M (on the cell surface of most serotypes) 
virulence fc
• SPE (Streptoccocal Pyrogenic Exotoxins)
• Type A --- similar molecule as Staph TSST-1
•Type B
•Type C
 Diseases caused by
Group A Streptococcus

Weiss, 1996
Identification of group A streptococci
• Bacitracin Test
– Streak streptococci on MHA
– Place Bacitracin impregnated filter paper disc on
agar, incubate 24 hours
Group A: observe zone of growth inhibition
(=sensitive to bacitracin)

• Antigenic-antibody reaction
– Extract the specimen, react with latex particle
coated with antibody to streptococci, observe
agglutination
Identification of group B streptococci
CAMP substance is produced by group B
streptococci that works sinergically with strong
betahemolysis of Staphylococcus aureus

Streptococci is streaked on to Blood agar

plate, adjacent to (at a right angle) to a
line streaking of Staphylococcus aureus (2
mm distance), incubate 37oC
If it is a Group B streptococcus: an
arrowhead zone of increased hemolysis,
other group shows normal hemolisis
around colony
 CAMP TEST: Lytic phenomenon
• S. agalactiae on CAMP test shows increased
hemolysis area when is near to S.aureus

1. Enterococcus faecalis
2. Streptococcus salivarius
3. Streptococcus agalactiae
4. Enterococus durans

Reference: Christie, R., N. E. Atkins, and E. Munch-Peterson. 1944. A note on a lytic

phenomenon shown by group B streptococci. Aust. J. Exp. Biol. Med. Sci. 22:197-200.
Identification of group D streptococci
• Bile esculin test pos:
streptococcus group D and enterococci
hydrolyses esculin into
6,7dihidroxycumarin, resulting brownish
black on the bile esculin medium.
– Neg esculin test : Viridans
group streptococci Enterococcus faecalis positive
Streptococcus mitis negative
• Grows in 6.5% NaCl broth
(broth turns cloudy).
Other groups do not.
 S.E.M of Streptococcus gallolyticus
(Group D Streptococcus)

After overnight incubation in modified BHI. Bar = 1 uM

L. O’Donovan, 2001
 REVIEW ON Staphylococcus
• Morphology:
– Colony: circle, white opaque-yellow
– Microscopic: irregular clusters, Coccus, gram
positive
• Major species:
– Staphylococcus aureus
– Staphylococcus saphrophyticus & S. epidermidis
(normally avirulent, sometimes cause skin lesions
and endocarditis)
Diseases caused by Staphylococci:
• Abscesses or minor skin inflammation
• Pneumoniae
• Osteomyelitis
• Endocarditis
• Cystitis
• Pyelonephritis
• Food intoxication
• Toxic shock syndrome Toxin-1 (TSST-1)
• Staphylococcus scalded skin syndrome (SSSS)
• Septicemia
• The genus comprises 50 taxons with 39
various types, and several subtypes
• Resistant to adverse environmental
conditions
• Resist drying
• Resist high NaCl concentration
• S. aureus is normal flora in anterior nares and perineum
 nasal mucosa carrier rate 37.2% (Ref.Matouska,
2008)
• S. epidermidis normal found in anterior nares anterior
and the skin
• S. saphrophyticus normal in the urinary tract
• Other Staphylococcus are common on other parts of the
human body
*all staphs may colonize cathether
*Among staphs only S. aureus produces exotoxins and able to
cause furuncles
*The exotoxins are the exfoliatin pyrogenic and superantigenik
toxins
 Metabolic end products of Staphylococci
1. Coagulase (cause clot formation)
2. Hyaluronidase (spreading factor)
3. Leukocidin toxin (makes pores that cause lysis of white
blood cell) : eg. Panton Valentine Leucocidin or Luk PV
which is produced by CA-MRSA
4. Haemolysin toxin
5. Staphylococcal superantigens (toxin)
6. Enterotoxin (exotoxins secreted by some strains of S.
aureus)
7. Dnase, lipase, gelatinase, penicillinase – non toxigenic
8. Staphylokinase -- fibrinolysin
 Identification of Staphylococcus
1. MSA test
– Agar medium containing Mannitol and high Salt
concentration
◦ S.aureus: yellow halo forms around the colony
2. Coagulase test
 Coagulase converts fibrinogen to fibrin
specimen which is mixed with citrated-plasma
results in coagulation = Coagulase positive
Absence of coagulase = Coagulase negative
• 3.Dioxyribonuklease test (DNase test)
– Agar medium contains DNA
– Specimen is spread on agar, hydrolisis of DNA by the
DNase is seen as pink halo (clear area around the colony);
– If DNase is not present, HCL reacts with DNA in the
medium and forms precipitation around the colony
• 4. Novobiocin sensitivity
– Able to distinguish :
• S. epidermidis from S. saphrophyticus
• Str. viridans from other streptoccocci
– Requires Mueller Hinton Agar and novobiocin disc
 Staphylococcus’ characteristics
– No flagella
– Non motile
– Non spore producing
• Aerob metabolism; can also undergo
facultative anaerob metabolism
• Distinguishing Streptococcus from Staph is by
Staph’s ability to produce catalase 
Streptococci are catalase and oxydase
negative and many are facultative anaerobe
 S. aureus’ cell membrane:
• composed of a combination of peptidoglycan and
teichoic-ribitol acid molecules, determines antigenicity
and relatively specific for S. aureus
• majority of S. aureus possess peptidoglycan covered by
a protein A.
• protein A uniquely binds Fc part of IgG molecule, thus
leaving only Fab part of IgG free to bind with antigen
 S. aureus becomes more virulence because of its
ability to deter opsonisation.
• (Opsonisation is the binding of antibody to antigen which then will
be swallowed by phagocytes)
 The growth of S. aureus
• Characteristic growth of S. aureus may be viewed
on medium containing 5% sheep’s blood  5 ml
of blood is added into 95ml autoclaved culture
medium at + 50oC poured into 5 sterile
petridishes
• Most S. aureus produces beta hemolysis around
its colony (complete hemolysis)
• After incubation overnight whitish colony is
formed with a tendency to turn to golden colour
HEMOLYSIS ß & NON HEMOLYSIS

On this agar cleared area can be Note that the agar medium remained
seen around the bacterial colony red because no lysis occurred
 Yellow colony diameter 2 mm
With hemolisis surrounding the colony

White colony

no hemolisis
 The difference of S. aureus as compared to
other Staphs:

• Coagulase binds prothrombin (non enzimatic

rection) and causes polimerization of fibrins
• COAGULASE /Slide clumping test
S. aureus + plasma clots fibrin (within hours)
or when concentrated emulsion of S.aureus +
plasma coagulates readily/very soon
S.epidermidis, S.lugdunensis  no coagulation
 Toxins of S. aureus
1. Alfa toxin
2. Exfoliatin
3. Pyrogenic Toxin Superantigen
 Alpha toxin
All S. aureus produce Alpha toxin, except when
it is one of a coagulase-negative strain.
Lyse sitoplasm membrane and form a
transmembrane pore (reviewed in Bhadki S, et
al. Alpha toxin of S. aureus, Microbiol Review,
1991;55: 733-751)
Alpha toxin acts similar with other cytolysins,
such as the Streptolisin-O, complements and
protein effector of cytotoxic T lymphocyte
 Exfoliatin
• Degrades intercellular bonds thus causing the
separation of epidermal layer between the
stratum spinosum and stratum granulosum
• Antigenic property:body produces antibody
against expholiatin
 Pyrogenic Toxin Superantigen
• PTSAg stimulates a systemic effect when
absorbed from a S. aureus infected site
• About 10% S. aureus cannot produce PTSAg
• One strain may produce one or more of the
Sag toxin
• Physically, chemically and biologically the
PTSAg of staphylococcus is similar with that
of streptococcus
 CoNS :
CoNS includes Staphylocococcus epidermidis, S.
lugdunensis, S. haemolyticus
Normal flora on human skin, but sometimes c/
illness
Infection is associated with cathethers, decreased
immunity, newborn, inplanted medical devices 
a causative agent
Multidrug resistant:
◦ R/ Vancomycin, Rifampin and newer quinolones
◦ Remove medical devices as source of infection
 CoNS
• May cause serious hospital infection
• foreign body-related infections
• infections in preterm newborns.
• S. saprophyticus acute urethritis
• S. lugdunensis causing infectious endocarditis.
• food-associated saprophytes colonizing the
skin and mucous membranes of humans and
animals
 Staphylococcus epidermidis
• Source of infection:
– Skin: from venous cath (IV or Hemodialysis) or
peritoneal dialysis
– From cathethers in the UTI
– Prosthetic joints
– Vascular grafts
– Eyes infection/surgery
– Other implants
 Virulence of Staphylococcus epidermidis

• Not much known

• Capsule or slime  able to form biofilm to
avoid elimination and cause infection from
invasive medical devices
 Staphylococcus epidermidis
• On solid culture (plate or slant agar): small
colony diameter ~ 1 µm (as compared to
S.aureus), white to cream
• Grows best in Blood agar: non hemolytic
• Grows on other non selective media
• Microscopy: Gram positive, single/in
pairs/short chains/in clusters
• A.k.a. Coagulase negative staphylococcus
 S.lugdunensis
Tube coagulase (long coagulase)
test. 
S. lugdunensis IDRL-5258 (top) is
unable to clot plasma
S. aureus RN4220 (bottom) clots
plasma

Ornithine decarboxylase test.  Ornithin

S. aureus RN4220 (left) Neg = yellow decarboxylase
S. lugdunensis IDRL-5258 (right) were pos = purple
used to inoculate in decarboxylase
medium agar
(Moeller decarboxylase broth base
containing ornithine and overlaid with
mineral oil.   Decarboxylase medium agar
pH indicator (bromocresol purple)
  2008 Jan; 21(1): 111–133.
Frank KL, Clin Microbiol Rev.
Rapid PYR test for S.lugdunensis
• S. aureus RN4220 (top) is PYR
negative.
• S. lugdunensis IDRL-5258
(bottom) produces PYR
(test pos)

Reading of result in
5 min after test

Frank KL, Clin Microbiol Rev. 2008 Jan; 21(1): 111–133.

 Synergistic hemolysis.
The δ-like hemolysin of S. lugdunensis,
acts synergistically with the β-hemolysin
of S. aureus to produce a zone of
complete hemolysis on sheep red blood
cells. 
S. lugdunensis IDRL-5258 (left side)
and S. lugdunensis IDRL-2414 (right,
streaked in triplicate) are streaked
perpendicular to, but not touching, S.
aureus RN4220, which is streaked
vertically.

photograph was taken 18 h after

inoculation and incubation in ambient air
at 37°C.

Frank KL, Clin Microbiol Rev. 2008 Jan; 21(1): 111–133.

Scanning electron micrograph of
S.lugdunensis

magnification ×11,000 of S. lugdunensis IDRL-2640 biofilm formed on a

silicone elastomer disk (Bentec Medical, Woodland, CA) after 24 h.

Ref: Frank KL, Clin Microbiol Rev. 2008 Jan; 21(1): 111–133.
 METHICILLIN RESISTANT
STAPHYLOCOCCUS AUREUS
• Methicillin-(and Oxacillin) resistant Staphylococcus
aureus (MRSA) are strains resistant to all β-lactam agents,
including cephalosporins and carbapenems.
• Pathogenic  Virulence factors enable them to result in
disease.
• Important c/ of nosocomial infections worldwide.
• Becomes Outbreak if one strain is transmitted to other
patients or through close contacts of infected persons in
the community.
– Hospital-associated MRSA (HA-MRSA) isolates are also
frequent causes of healthcare-associated bloodstream and
catheter-related infections.
– Community-associated MRSA (CA-MRSA) isolates are
often only resistant to beta-lactam agents and erythromycin
 References:
1. McPhee S.J. Et al in Current Medical Diagnosis and Treatment, 2011
2. Ruff CT et, al, in Hurst’s the Heart, Manual of Cardiology 12 ed, 2009
3. Manual of Cardiovascular Medicine, 3rd edition, Brian P Griffin and Eric J.
Topol, editors, Lippincott Williams and Wilkins, 2009.
4. Medical Microbiology, 3rd edition, Cedric Mims, et al (eds), Mosby, 2004.
5. Infectious Diseases, 2nd edition, Jonathan Cohen and William G. Powderly
(eds)
6. Zinsser Microbiology, 20th edition
7. Valvular Heart Disease, 3rd edition, Joseph S. Alpert, James E.Dalen,
Rahimtoola Shahbudin H., editors, Lippincott Williams and Wilkins, 2000
8. Bhadki S, etal. Alpha toxin of S. aureus, Microbiol Review, 1991;55: 733-
751.
9. Patophysiology of Heart Diseases, 3rd edition, Lilly LS. (Ed), Lippincott
Williams and Wilkins, 2003
10. Matouskova I, 2008, Current knowledge of MRSA and CA-MRSA, Biomed
Pap Med Fac Univ Palacky Otomouc Czech Repub, 2008, 152 (2): 191-202
11. Others as cited in this lecture slides.