AAU College of Health Sciences Microbiology and Parasitology Module Schedule

Addis Ababa University
College of Health Sciences
School of Medicine
Dept of Microbiology, Immunology and Parasitology
Afework Kassu, PhD
Professor of Microbiology and Immunology
afeworkkassu@yahoo.com
0921221959

Module 6 Schedule
Theme 4 - Microbiology & Parasitology. Week 6 & 7
January 28 - February 10, 2015
Date /Time Microbiology Lecture hrs Practical
4. Skin and soft tissue infections 13 hrs total 9 hrs Group Day
28/1/15
9.00-10:30
10:45-12.00
4.1. Streptococcal Infections
Staphylococcal infections
2 hrs
Prof Afework
3 2,5
3,6
1,4
Wed
Thur
Fri
29/1/15
9.00-10:30
10:45-12.00
4.2 - Gas-gangrene
4.3 - Leprosy
2 hrs
Dr Solomon
30/1/15
9.00-10:30
10:45-12.00
4.4 - Viral exantems
Viral exantems
2 hrs
Prof Afework
2/2/15
9.00-10:30
10:45-12.00
Test III
4.5 - Bone & joint microbial disease
 Osteomyelistis
1 hr
Dr Daniel
3/2/15
9.00-10:30
10:45-12.00
 Arthritis
4.6 - Superficial mycosis
1 hr
Dr Daniel
Dr Yimtu (1 hr)
4/2/15
9.00-10:30
10:45-12.00
4.7 - Cutaneous mycosis
4.8 - Opportunistic fungal infections
2 hrs
Dr Yimtu
3 1, 4
2,5
3,6
5/2/15
9.00-10:30
10.45-12:00
4.9 - Scabiasis
4.10 – Pediculosis
2 hr
Tadesse
6/2/15
9.00-10:30
10:45-12.00
4.11 - Cutaneous Onchocerciasis
1 hrs
Tadesse
10/2/15
10:45-12.00 4.12 - Lesimaniasis
1 hr
Prof Asrat
3 3,6/1,4
2,5
Feb 23-27 Final Module exam (Test IV) Feb 23-27

Theme 4. Lectures and seminars
Skin and soft tissue infections
• Staphylococci and streptococci
• Leprosy
• Superficial mycosis
• Cutaneous Mycoses
• Opportunistic fungal infections
• Viral exantem
• Gas-gangrene
-- Scabiasis
-- Pediculosis
• Cutaneous Onchocerciasis and Leishmaniasis
• Bone and Joint microbial diseases - Osteomyelitis
- Arthritis

Theme 4. Microbiology Practical Classes
• Streptococcal & staphylococcal infection (Impetigo, erysipelas)
• Leprosy
• Superficial mycosis
• Cutaneous mycosis
• Subcutaneous mycosis
• Opportunistic fungal infections
• Viral exantem (small pox, herpesviruses, parvoviruses,
papiloma virus)
• Gas-gangrene
• Scabiasis
• Pediculosis
• Cutaneous Onchocerciasis and Leishmaniasis

References
• Jawetz Medical Microbiology
• Murray P.R. Rosenthal K.S, Kobayashi G.S,
Pfaller M.A. Medical Microbiology
• Robert Boyd. Basic Medical Microbiology
• Others

Staphylococcal and streptococcal
infections
Objectives:
Upon completion of this section the student will
be able to:
• Discuss the basic characteristics of
staphyloccocci and streptococci
• Describe their virulence factors
• Discuss pathogenicity, clinical
manifestations, laboratory diagnosis,
prevention & control

Staphylococcal and streptococcal infections

Staphylococcus
• Make very large contribution to human commensal flora
• Account for a high proportion of acute and chronic lesions.
• General properties/characteristics of staphylococci
– Gram-positive cocci of uniform size (1m in diameter).
– Arranged in Grape like clusters (but also found single or in
pairs), Staphyle, meaning ‘bunch of grapes’
– Non-motile,
– Non-spore forming
– Aerobic or facultative anaerobes
– Produce catalase
– Resistant to temp as high as 50°C,
drying, high salt concentration

Staphylococci of medical importance
• There are over 30 species of Staphylococcus
• Species of medical importance:
– Staphylococcus aureus
– S. epidermidis
– S. Saprophyticus

Staphylococcus aureus
• S. aureus most important human pathogen than the
other species of staphylococci.
• Is distinguished from the other species by:
- coagulase production
- manitol fermentation and
- hemolysis of RBCs (beta-haemolysis)
 More than 90% of S. aureus strains contain plasmids
that encode  -lactamases

Cultural characters:
• Facultative anaerobe
• Grow on nutrient agar producing golden yellow colonies of
1-2 mm (as a result of the carotenoid pigment that form
during growth, hence the species name)
• They produce β-hemolytic colonies on blood agar
Beta hemolysis

Biochemical features
• Fermentation of glucose produces mainly lactic
acid
• It ferments mannitol (distinguishes from S.
epidermidis)

Pathogenesis
 S. aureus produce disease b/c:
its ability to adhere the cells
spread in tissues & form abscesses,
produce extracellular enzymes and
exotoxins.

• S. aureus has several important cell wall components & antigens
- important virulence factors for S. aureus:
1. Structural components
a) Capsule or polysaccharide slime layer
b) Peptidoglycan
c) Teichoic acid
d) Protein A
2. Toxins
a) Cytotoxins
b) Exfoliative toxins
c) Enterotoxins
d) Toxic shock syndrome toxin-1
3. Enzymes: Coagulase, catalase, hyaluronidase, fibrinolysin,
lipases, nucleases & penicillinase

Fig . Virulence determinants of Staphylococcus aureus

• 1. Structural components
a) Capsule:
- inhibiting the chemotaxis & phagocytosis.
- facilitate attachment to catheters & other synthetic materials.
b) Peptidoglycan:
- provide osmotic stability,
- stimulates production of endogenous pyrogen (endotoxin like
activity),
- leukocytic chemo-attractant (abscess formation); and
- inhibit phagocytosis.
c) Teichoic acid:
-regulates cationic concentration at cell membrane and
-mediate adherence of the organism to mucosal cells.
d) Protein A:
- It binds to the Fc portion of IgG at the complement binding site,
there by preventing the activation of complement

2. Toxins
a) Cytotoxins: consisting of cytolytic or membrane damaging toxins
(alpha, beta, delta, gamma, leukocidin)
b) Exotoxins: three clinically important exotoxins
i) Enterotoxins: (A-E, G-I) Heat stable (resistant 1000c/30min.),
-unaffected by GI enzymes and are a cause of food poisoning,
principally associated with vomiting.
ii) Exfoliative toxin: cause desquamation of skin or scalded skin
syndrome in young children
 Serine protease (affect GM4-glycolipids which is more in
children than adults) is produced, which can split the
intercellular bridge in the stratum of granular cell layer that
leads to epedermolysis.
iii) TSST: common in tampon using manstruating women or
individual with wound infection.

 Staphylococcal enterotoxins, TSSTs and exfoliative
toxin are ‘super antigens’, all bind non-specifically to
specific white cells resulting in over production of
cytokines giving rise to a toxic shock-like
presentation.
• Diseases Associated with Superantigen production:
Toxic shock syndrome, psoriasis, rheumatoid arthritis,
Diabetes mellitus, Scarlet fever, Eczema

c) Enzymes:
• Coagulase: causes the clumping of non-capsulated strains
when mixed with a solution containing fibrinogen, e.g.
Plasma. Helps encase infection by forming fibrin layer
around abscess
• Catalase: Reduces phagocytic killing by converting H2O2 to
H2O
• Hyaluronidase: Spreading factors
• Fibrinolysin:
• Lipases:
• Nucleases:
• Penicillinase: Disrupts the Beta -lactame rings

Factors Biological action
Surface components
Capsule Antiphagocytic
Protein A Inhibits complement fixation, opsonization, and ADCC
Teichoic acid and
lipoteichoic acid
Promote adherence to mucosal surfaces and persistence in tissues by
binding to fibronectin
Enzymes
Catalase Reduces phagocytic killing by converting H2O2 to H2O
Coagulase Helps encase infection by forming fibrin layer around abscess
Degradiative enzymes Promote tissue destruction and bacterial spread
Beta lactamase Confers antibiotic resistance
Toxins
Leukocidins Damage and lyse leukocytes; releases tissue-damaging substances
Enterotoxins Act as superantigens; responsible for gastrointestinal food poisoning
Exfolative toxins Cause splitting of cell junctions in epidermis; responsible for scalded
skin syndrome
Toxic shock syndrome
toxin
Acts as superantigen; promotes massive cytokine release, causes
toxic shock syndrome

Diseases
The important clinical manifestations caused by S.aureus can
be divided in to two groups:
1. Inflammatory diseases :
- Superficial/skin infection (Folliculitis, carbuncles, boils, stye, mastitis,
abscess formation, impetigo, furuncles, cellulites, surgical
wound infections and mastitis) and
- Deep-seated/systemic diseases (Osteomyelitis, septic arthritis,
endocarditis, meningitis, bronchopneumonia, empyema, etc).
- Bacteraemia with multiple abscesses in tissues: Outbreaks of
hospital wound infections commonly occur due to antibiotic
resistant staphylococci.
2. Toxin medicated diseases:
 These include food poisoning, toxic shock syndrome, and
scalded skin syndrome.

B. Toxin-mediated staphylococcal diseases
1. Food poisoning
– Results from ingestion of preformed
enterotoxin in contaminated food that is
improperly cooked and kept unrefrigerated for
some time.
– Source of contamination of food: the hands or
nose of a cook / food handlers /carriers.
– Types of food involved in staphylococcal food
poisoning are carbohydrate rich foods, e.g.
cakes, pastry, milk, etc.
– IP: short (1-8 hrs) followed by nausea, vomiting,
diarrhoea and general malaise with no fever.
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2. Toxic shock syndrome (TSS):
– This is associated with TSST-1, first described
in menstruating women using tampons. The
syndrome also occurs with wound or localized
infections.
– TSS has an abrupt onset of fever, vomiting,
diarrhoea, muscle pains, rash
– Hypotension, heart and renal failure may occur
in severe cases.
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F I G U R E 1 6 – 3
Pathogenesis of staphylococcal toxic shock syndrome. A. The vagina is
colonized with normal flora
and a strain of Staphylococcus aureus containing the TSST-1 gene. B. The
conditions with tampon
usage facilitate growth of the S. aureus and TSST-1 production. C. The toxin
is absorbed from the
vagina and circulates. The systemic effects may be due to the direct effect of
the toxin or via cytokines
released by the superantigen mechanism. The toxin is shown binding
directly with the V
portion of the T-cell receptor and the class II major histocompatibility
complex (MHC) receptor.
This V stimulation signals the production of cytokines such as interleukin-1
(IL-1) and tumor
necrosis factor (TNF).

3. Staphylococcal scalded skin syndrome (SSSS):
– Occurs due to the exfoliative toxin produced by
phage II strains of S. aureus.
– The syndrome occurs in babies and young
children.
– It is characterized by large areas of
desquamation of the skin and generalized
bullae formation.
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FIGURE . Evidence of staphylococcal scalded-skin syndrome in a 6-year-old boy.
Nikolsky’s sign, with separation of the superficial layer of the outer epidermal
layer, is visible.

(Richard A. Harvey, Pamella C. Champ, Microbiology, Lippincott’s illustrated reviews, 2nd ed.)

• Epidemiology
• It is normal flora on human skin, nose (25 – 75%
carriers) and mucosal surfaces
• The organism can survive on dry surfaces for long periods
• Transmission is from person to person through
direct contact or exposure to contaminated fomites
(e.g. bed linens, clothing)
• Risk factors include presence of a foreign body such
as suture and catheter, previous surgical procedure
and use of immuno-suppresser drugs
• Patients at risk for specific disease include:-
menstruating women,
 infants ,
young children with poor personal hygiene;
 patients with compromised pulmonary function

Laboratory diagnosis:
Specimens
– Swabs from lesions: pus, sputum, CSF, blood, urine examined
Gram stain smears: Gram positive cocci in clusters
Culture:
– S. aureus colonies show complete haemolysis on blood
agar and golden yellow to cream & occasionally white
1-2 mm in diameter colonies on nutrient agar.
– Colonies are usually large (6-8 mm in diameter),
smooth & translucent.
– Mannitol salt agar (MSA) - useful differential and
selective medium for recovering S. aureus from fecal
specimens in staphylococcal food-poisoning.
30

Isolated colonies are further identified by:
1. Catalase test: Staphylococci are catalase positive
2. Cougulase test: tested by adding few drops of a broth
culture of the organism to 0.5ml of human or rabbit
plasma; coagulation of the plasma occurs within few min.
3. Phage typing:
– used for epidemiologic tracing of "out-breaks of
wound infections" in hospitals. S. aureus isolated from
wounds, nose, nail bed of attending Drs, nurses and
families should be identified by phage typing to reveal
the source of infection
– In tracing the source of contamination of food in ‘out-
breaks of food poisoning’; the organisms isolated
from food, vomitus, stool, nose & nail bed of food
handlers are phage typed.
– Most enterotoxin-producing strains of S. aureus
belong to phage group III.
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Antimicrobial sensitivity
– Several antibiotics are effective against staphylococci
e.g. pen, erythromycin, tetracyclines, ampicillin,
cephalosporins.
– S. aureus shows marked ability to develop resistance to
antibiotics specially in hospitals.
– Sensitivity testing is therefore essential for the choice
of the appropriate antibiotic.
– About 80% of hospital strains of staphylococci are
penicillin resistant. This is due to the production of β-
lactamase which breaks down the lactam ring in
penicillin & cephalosporins
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Treatment of S. aureus infections
• For penicillin / methicillin sensitive organisms – Penicillin or
cephalosporin
• TMP-SMX, rifampicin, Clindamycin, Quinolone
• Some recommend combination therapy - Amoxicillin &
clavulanate (Augmentin)
Drug Resistance
• Methicillin-resistant S. aureus (MRSA) - (esp in hospital
settings)
– Recently, vancomycin resistant S aureus emerging
– CA-MRSA (community-acquired–MRSA) cause about half
of the cases
– HA-MRSA (Healthcare-Associated-MRSA)
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Hemolysis of BAP
alpha hemolysisBeta hemolysis

• They are coagulase-negative staphylococci
- Do not produce exotoxins.
- Thus, they do not cause food poisoning or toxic shock
syndrome.
S. epidermidis
- Part of the normal flora of the skin and mucous membrane.
- Cause of hospital-acquired infections
- It is involved in indwelling catheters, prosthetic materials,
shunts, surgery
- It is a common cause of prosthetic heart valve endocarditis
- It also causes nosocomial bacteremia; sepsis in neonates
peritonitis in patients with renal failure; cerebrospinal fluid
shunt infections
- Often multiple antibiotic resistance - Methicllin
S. epidermidis & S. saprophyticus

S. saprophyticus
• Commonly isolated from animals and their carcasses.
• Coagulase and phosphatase -ve, urease & Lipase +ve
• S. saprophyticus is resistant to the antibiotic Novobiocin, a
characteristic used in lab to distinguish it from S. epidermidis
• Infections are almost always community acquired.
• It causes mainly UTI, particularly in sexually active young
women.
• It is a 2nd most common cause of UTI, after E. coli in young
women accounting for 10-20% .
• It also causes soft tissue infections.
• Quinolones are commonly used in treatment of S. saprophyticus
UTI.

Table: Biochemical characteristic of
staphylococci

Fig. Flow diagram for
the preliminary
identification of human
Staphylococcus species

General characteristics:
• Non-motile, non-spore forming, catalase-negative
Gram positive cocci arranged in chains or pairs.
• Widely distributed in nature, some are commensals in
the throat, intestine.
• Others cause human diseases
• Others found in water, dust, milk /milk products.
• Most are facultative anaerobes and some grow only in
an atmosphere enhanced with carbondioxide.
• Elaborate variety of extracellular substances &
enzymes
• Their nutritional requirements are complex
necessitating the use of blood or serum-enriched
media for isolation
• More than 30 spp are identified.
Streptococcus

Streptococcus
• Scanning electron microscope image
of Streptococcus
2/9/2015 42

Streptococcus
Classification:
• Heterogenous group of bacteria
I. According to their O2 requirements they are either aerobic or
anaerobic (peptostreptococci).
A. Aerobic streptococci according to their action on RBC in blood
agar (3 varieties):
1. Beta haemolytic streptococci: complete haemolysis (clear
zone around the colonies on BA) e.g. S. pyogenes, S.
agalactiae
2. Alpha haemolytic streptococci: partial haemolysis
e.g. Viridans streptococci, S. pneumoniae
3. Non-haemolytic or gamma streptococci: no haemolysis/
change in RBC's, e.g. Enterococci
2/9/2015 43

Beta haemolysis
alpha-
haemolysis)
Gamma haemolysis
Streptococcus

Streptococcus
II. Classification based on serologic reactivity of cell wall
polysaccharide Ags as originally described by R. Lancefield
• >18 group-specific Ags (Lancefield groups A-U) were
established according to the carbohydrate (C) Ag present in
the cell wall.
• Group A: S. Pyogenes - causes pharyngitis, skin infections
• Group B: S agalactiae - causes neonatal septicemia,
meningitis
• Group C: S. equisimilis – endocarditis, bactermia, meningitis
• Group D: Enterococci – UTI, endocarditis
• Group H: S. sanguis – endocarditis, dental caries
• Group K: S. salivarius - endocarditis, caries
2/9/2015 45

III. Classification based on Biochemical (physiologic)
properties:
- some streptococciare difficult to classify by
hemolytic & antigenic xics
- biochemical tests are used in their final
classification.
Streptococcus

Streptococcus pyogenes
Group A streptococcus (GAS): Streptococcus pyogenes
• GAS consists of 1 spp with 40 antigenic types
• S. pyogenes, the most important human pathogen
causing diseases including:
– Suppurative conditions / Skin infections
– Throat infections
– Systemic infections
– Non-suppurative sequelae
• About 5-15% of normal individuals harbor the
bacterium, in their respiratory tract, without signs of
disease.
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Morphology:
• Gram-positive cocci arranged in chains of varying length &
are non motile.
• Some strains are capsulated.
• Forms minute colonies
• It grows on blood agar producing β-haemolysis
Antigens
• The cytoplasmic membrane of S. pyogenes has Ags similar
to those of human cardiac, skeletal, smooth muscle, heart
valve fibroblasts and neuronal tissues, resulting in a
molecular mimicry.
2/9/2015 50

Virulence factors of group A streptococci:
1. M – protein, fibronectin-binding proteins (e.g. F
protein) & lipoteichoic acid for attachment
2. Hyaluronic acid capsule: inhibits phagocytosis
3. Exotoxins such as pyrogenic toxin, cause the rash
of scarlet fever & systemic toxic shock syndrome
4. Invasins - Streptokinase, streptodornase (DNase
B), hyaluronidase & streptolysins
2/9/2015 51

Figure: Antigenic structure of S. pyogenes and adhesion to an epithelial cell. The location of peptidoglycan
and Lancefield carbohydrate antigen in the cell wall is shown in the diagram. M protein and lipoteichoic
acid are associated with the cell surface and the pili. Lipoteichoic acid and protein F mediate
binding to fibronectin on the host surface.

Protein antigens:
• S. pyogenes: divided into types based on their content of M
protein (about 80 types described).
• M protein:
- the most important virulence factor
– is antiphagocytic
- promotes adherence to host epithelial cells
• The protein and other cell wall Ags have important role in the
pathogenesis of rheumatic fever.
• Antibodies to these components react with cardiac muscle
tissue.
• Other antigens are the T & R proteins - but they are not
related to virulence.
2/9/2015 53

Toxins and Enzymes:
• More than 20 extracellular products that are antigenic are
produced by S. Pyogenes.
Some of the important toxins and enzymes include:
1. Streptokinase (fibrinolysin): It activates plasmin in blood,
which can dissolve fibrin in clots, thrombi and emboli, causes
fibrinolysis.
2. Streptodornase (deoxyribonuclease): DNAses (types A, B, C, D)
that break down DNA and stimulate an antibody response
especially against DNAse B.
3. Streptokinase and streptodornase are responsible for the
spreading nature of streptococcal infections.
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4. Hyaluronidase:
– It destroys hyaluronic acid, the cement substance of
connective tissue. It helps spreading of infection.
5. Streptococcal pyrogenic exotoxins (SPE) (formerly
erythrogenic toxin):
– An exotoxin produced by some strains of group A
streptococci, lysogenized by a bacteriophage carrying the
gene for the toxin,
– Act as superantigens
– It causes vasodilatation of capillaries leading to the rash
of scarlet fever.
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6. Streptolysins: There are 2 types:
a. Streptolysin O - (O2 labile):
– Antigenic, stimulates the production of
antistreptolysin O (ASO) Ab that can be measured in
patients serum.
b. Streptolysin S - (O2 stable):
– Not antigenic, does not stimulate production of Ab
– It hemolyzes red cells and is responsible for the beta -
haemolysis produced on blood agar.
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Pathogenesis:
I. Diseases due to toxins
• S. pyogenes: exogenous secondary invader, following
viral dis or disturbances in the normal bacterial flora
1. Streptococcal sore throat and follicular tonsillitis
(pharyngitis): It is characterized by enlarged tonsils with purulent
exudate, high fever and enlarged cervical lymph nodes; the most
common infection caused.
2. Impetigo: A local infection of the skin characterized by
formation of blisters which break leaving surface covered
with pus or crusts
3. Scarlet fever: disease of children characterized by sore
throat and erythematous rash; caused by erythrogenic toxin
in susceptible individuals.
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II. Diseases due to invasion:
– Diffuse rapidly spreading infections that involve the
lymphatics with minimal local suppuration infection can
extend to the blood stream.
1. Puerperal sepsis: Infection of the uterus after delivery or
abortion leading to endometritis and is associated with
bacteremia.
2. Erysipelas: a skin infection characterized by redness,
edema and a rapidly advancing margin
3. Soft tissue sepsis: Wound infection, cellulitis,
lymphadenitis, necrotizing fascitis - may be complicated by
streptococcal septicemia.
4. Acute Bacterial Endocarditis: The organism reaches the
heart valve through the blood stream as a complication of any
of the primary lesions mentioned before.
– The presence of a deformed or rheumatically affected
valve encourages the condition.
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III. NONSUPPURATIVE SEQUELAE:
Post-streptococcal diseases –GN, RF
• May occur 1- 4 weeks following a primary inadequately treated
group A streptococcal infection of the skin & RT
• The latent period suggests hypersensitivity to streptococcal products
is the most probable cause.
• GN- may follow throat or skin infections (pyoderma) while
rheumatic fever follows only throat infections (pharyngitis).
1. Acute glomerulonephritis (AGN):
• develops 3 weeks after infection with a nephritogenic strain of
streptococci (types 12, 4, 2 & 49).
• Disease is characterized by fever, edema, elevated BUN (azotemia),
hematuria, high BP, low serum complement levels. Blood, albumin,
granular casts present in urine.
• The condition is due to Ag-Ab complex deposition on the glomerular
basement membrane (GBM).
• The majority of patients recover completely. However few may die
or pass to chronic GN & renal failure.
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2. Rheumatic fever (RF):
• It has a more tendency to recur than GN because RF can
result from infection by any of the serotypes of S. pyogenes
where as GN is associated with only a limited number of
these serotypes.
• The onset follows 1-4 weeks after throat infection with
group A streptococci.
• The most serious complication of streptococcal throat
infection since it may result in damage of the heart valves &
muscle.
• RF is characterized by fever, migrating polyarthritis,
carditis, Erythema marginatum, Sydenham chorea
2/9/2015 60

• The pathogenesis of RF is an autoimmune disease.
• Streptococci have Ags immunologically similar to
proteins present in the heart valves and muscle, so
the Abs produced against streptococci can react
with the heart (i.e. cross reactivity) leading to
cardiac damage.
• Recurrence of rheumatic activity occurs due to
repeated streptococcal infections and every attack
adds to the cardiac damage.
• This can be prevented by prophylactic long acting
penicillin administration.
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Epidemiology
• 5-15% of humans carry S. pyogenes or S. agalactiae in the
nasopharynx.
• Vehicles: foods, like cow’s milk, egg or potato salad
• Infective dose: <1000 cells
• Onset of illness occur within 1-3 days
• Spread by respiratory droplets or by contact with fomites used
by the index individual, either patient or carrier
• Skin infections often follow minor skin irritation, such as
insect bites.
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Lab diagnosis of streptococcal diseases:
1. Specimens: Swabs from throat or other lesions, pus,
or blood in case of bacteraemia
2. Direct smears stained by Gram’s method show Gram-
positive cocci in chains.
3. Cultures done on blood agar show colonies producing
complete haemolysis
4. Blood cultures: done for bacteremic infections e.g.
bacterial endocarditis & puerperal sepsis. In the latter
case, it is more valuable in diagnosis than the uterine
swab, which is often contaminated with normal flora.
2/9/2015 63

5. Bacitracin test:
• GAS can be differentiated from other other β-hemolytic
streptococci (B, C, G) by their sensitivity to bacitracin.
• The test is done by placing a filter paper disc containing
bacitracin (0.05um) on blood agar inoculated with the
organism. A zone of inhibition around the disc observed
6. Antigen detection tests:
• Rapid detection of group A streptococcal Ags from throat
swab can be done by ELISA or agglutination tests
2/9/2015 64

7. Serologic tests – for anti-streptococcal antibody titers
• mainly used for diagnosis of post streptococcal diseases-
Acute GN and acute RF identification
a. Antistreptolysin O (ASO): Abs develop in pt sera with
streptococcal infections. Detection of high ASO titre of 1/200
or more is significant.
b. C-reactive protein (CRP):
• CRP: abnormal protein that appears in serum in cases of
active RF, other degenerative & inflammatory conditions
• It can be tested for by a passive agglutination reaction using
latex particles coated with anti-CRP Abs
• A positive CRP, a high ASO titre and high ESR are helpful in
the diagnosis and follow up of cases of RF
2/9/2015 65

Treatment and Chemoprophylaxis:
• Penicillin is the drug of choice for treatment of
streptococcal diseases.
• Long acting penicillins: given as chemo-
prophylactic measure to children prone to
recurrent attacks of streptococcal sore throat or
recurrent attacks of RF.
• Erythromycin is also effective, BUT Erythromycin
resistance (check MICs by Etest)
• Macrolide resistance in ß-haemolytic streptococci
of Lancefield groups A, B, C, G
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Streptococcus agalactiae
Group B streptococci (GBS): S. agalactiae
• About 10% - 30% of pregnant women are colonized with GBS
in the genital tract.
• GBS causes life-threatening infections in newborn infants
• GBS can also cause serious diseases in pregnant women, the
elderly, and adults with other illnesses.
• In newborns, GBS is the most common cause of sepsis and
meningitis and a common cause of pneumonia.
• GBS disease in newborns usually occurs in the first week of
life.
2/9/2015 68

• Babies can also get a slightly less serious "late-onset" form
of GBS disease that develops a week to a few months after
birth.
• Adults with illnesses that weaken the immune system, such
as diabetes or cancer, are at risk of infection with GBS.
Diagnosis
• Lab test of blood or spinal fluid.
• Group B streptococci show hippurate hydrolysis
Treatment
• Newborns and adults are usually treated with antibiotics
given IV
2/9/2015 69

Prevention
• Any pregnant woman who has already had a baby
with GBS infection or who has a urinary tract
infection caused by GBS should be given antibiotics
during labor.
• Pregnant women who are colonized with GBS should
be offered antibiotics at the time of labor or rupture
of the membranes.
2/9/2015 70

Viridans Streptococci
Alpha Haemolytic Streptococci – Group D
Viridans Streptococci Group
• Non-pyogenic streptococci
• Are biochemically & antigenically diverse group
• They form part of the normal flora of the mouth.
• They lack either the polysaccharide-based capsule typical
of S. pneumoniae or the Lancefield Ags of the pyogenic
members of the genus.
• Group D is differentiated from other viridans streptococci
by bile solubility and optochin sensitivity.
2/9/2015 72

Viridans and other species
• S. mutans, a contributor to dental caries. It synthesize
large sticky polysaccharide such as dextrans or levans from
sucrose and participate in dental carries production.
• S. mutans & S. sanguis - odontopathogens responsible for
the formation of dental plaque.
• S. mitis, mostly found around cheek region
• S. sanguinis, no preference of locations
• S. viridans, a cause of endocarditis, dental abscesses
• S. salivarius, found on the dorsal side of the tongue
• S. salivarius ssp. thermophilus, used in the manufacture of
some cheeses and yogurts
• S. constellatus, occasional human pathogen
2/9/2015 73

Diseases caused by Viridans streptococci:
• Cause sub-acute bacterial endocarditis (SBE) in individuals
with congenitally deformed or rheumatically affected heart
valves.
• Account for 30 - 40% of cases of endocarditis
• Infection is endogenous: organisms reach the blood stream
during tooth extraction or tonsillectomy.
• They settle on the deformed valve and lead to the
inflammatory process.
• Mortality: >50%
2/9/2015 74

Lab Diagnosis
Blood culture
• Add 5 -10ml of blood to 50 -100ml of broth, incubated at
37oC.
• Sub-cultures made on BA every 48 hrs for 10 days.
• The large volume of broth has the following advantages:
– Dilutes out antibacterial substance naturally occurring in
serum.
– Allows for multiplication of organisms present in few
numbers.
– Antagonists to antibiotics taken by the patient can be
added to the broth
2/9/2015 75

Enterococcus
Group D: Non-Haemolytic Streptococci
1. Enterococcus
• >12 spp, some of these are - Enterococcus faecalis, E.
fecium
• They are normal inhabitants of the intestine.
• They cause UTI, wound, cholecystitis, blood infections,
meningitis, bacteremia (neonates) subacute endocarditis,
prostatitis.
• Enterococci are among the most common causes of
nosocomial infections, esp in ICU
2/9/2015 77

Enterococcus
• E. faecium causes 5-10% of enteroccal disease
• They are more resistant to antibiotics than other
streptococci.
• They show intrinsic resistance to cephalosporins,
monobactams, some penicillins.
• They show vancomycin resistance (VRE)
• Combined penicillin and aminoglycosides are used for
treatment.
2. Non-enterococcal Group D strains
• include Streptococcus bovis and Streptococcus equinus.
2/9/2015 78

PEPTOSTREPTOCOCCI
ANAEROBIC STREPTOCOCCI
PEPTOSTREPTOCOCCI
• Grow under anaerobic or microaerophilic conditions.
• They are normal inhabitants of the mouth, intestine,
URT & female genital tract.
• They are involved in serious mixed anaerobic
infections in the abdomen (abscesses), pelvis, lung,
brain, the female genital tract and pulmonary tract.
2/9/2015 79

STREPTOCOCCUS PNEUMONIAE
/Pneumococcus/
2/9/2015 80

STREPTOCOCCUS PNEUMONIAE
/Pneumococcus/
Habitat:
• S. pneumoniae is a normal inhabitant of the human upper
respiratory tract (found as commensal)
• Virulent pneumococci are often carried in the normal
respiratory tract of healthy people.
• The carrier rate of S. pneumoniae in the normal human
nasopharynx is 20-40%.
Morphology:
• Gram-positive, catalase-negative, lancet-shaped slightly
elongated and arranged in pairs, non-motile and non-
sporulating diplococci.
• Are capsulated in animal tissues.
• Capsules may appear as unstained halos around the organism.
• They do not display M protein
2/9/2015 81

Pneumococcus
Biochemical characters:
• Ferment glucose to lactic acid
• Doubling time is 20-30 min.
• Contain autolysin (enzyme) – which disrupts and
disintegrates the cells
• Most strains of S. pneumoniae are α-hemolytic but
can cause β-hemolysis during anaerobic incubation
2/9/2015 82

Pneumococcus
Pneumococci Viridans Strept
Solubility in bile Soluble Not soluble
Fermentation of
inulin
Fermented with acid
production
Not fermented
Sensitivity to
optochin
Sensitive Not sensitive
Pathogenicity to
mice
Pathogenic, cause
death of mice on
intraperit. injection
Not pathogenic
Quellung test Positive Negative
2/9/2015 83
- They grow on blood agar producing α-hemolysis similar to that
of viridans streptococci from which they are differentiated by the
following tests:

Pneumococcus
Antigenic structure:
• About 90 serotypes of pneumococci based on the chemical
specificity of the capsular polysaccharide.
• Serotyping can be done by agglutination or capsule swelling
"quellung reaction".When pneumococci of a certain type are
mixed with a specific anti-polysaccharide serum of the same
type or with a polyvalent antisera on a microscopic slide the
capsule swells markedly.
• This is useful for rapid identification and typing the organism
either in sputum or in culture.
• No Lancefield group antigen
2/9/2015 84

Pneumococcus
Pathogenesis:
• S. pneumoniae does not produce toxins
• It owes its virulence to the capsule, which enables the
organism to invade the tissues and resist phagocytosis.
• Predisposition to disease: if resistance is lowered, e.g. by viral
respiratory infections, excessive smoking, alcoholism,
malnutrition
Diseases caused by S. pneumoniae
• S. pneumoniae is currently the leading cause of invasive
bacterial disease in children and the elderly.
• Causes 80% of lobar pneumonia; conjunctivitis, paranasal
sinusitis, OM, meningitis, acute exacerbation of chronic
bronchitis, septic arthritis, osteomyelitis, endocarditis,
peritonitis, cellulitis, brain abscess.
2/9/2015 85

Pneumococcus
Laboratory diagnosis of lobar pneumonia:
1. Direct microscopic examination of gram stained sputum
smears
2. Sputum cultured on blood agar.
– Pneumococci produce α-haemolytic colonies, which should
be differentiated from viridans streptococci.
– S. pneumoniae is a fastidious bacterium, growing best in 5%
CO2.
– Nearly 20% of fresh clinical isolates require fully anaerobic
conditions.
2/9/2015 86

Pneumococcus
3. Quellung reaction: Fresh emulsified sputum mixed with
polyvalent anti-pneumococcal serum, stained by methylene
blue and examined under the microscope will show a
positive ‘quellung reaction’. This is done for rapid
identification.
4. Intraperitoneal injection of sputum into mice. The animal
dies in 24-48 hrs. The organism can be seen in tissue smears
& pure culture can be obtained from heart blood.
Treatment
• Pneumococci are sensitive to many antibiotics.
• Some isolates have recently been reported to be resistant to
penicillin.
2/9/2015 87

Pneumococcus
Prophylaxis / Prevention:
• 2 vaccines available to prevent pneumococcal
disease
1. Pneumococcal conjugate vaccine (PCV7)
– for infant immunization children,
2. Polyvalent polysaccharide vaccine (PPV23)
– for adults - are safe and fairly effective.
– The later is recommended for specially susceptible
individuals, e.g. aged (>65 years), debilitated or bed-
ridden patients or after splenectomy.
2/9/2015 88

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