Gram-Positive Cocci

Zeb Hussain,
BSc,(Biochem)BS,(MLT-CLS)M.Phil,(Pathology) E. MBA, PhD

ZEB 1
 CHAPTER CONTENTS

• Introduction
• Staphylococcus
• Streptococcus CHAPTER
15
• Streptococcus pneumonia
• Self-Assessment Questions
• Summaries of Organism
 Reference:

• Book name:

• Medical Microbiology and immunology 14th edition

• Chapter number – 15

• Page no: 109-127

 INTRODUCTION
• There are two medically important genera of gram-positive
cocci:
• Staphylococcus
• Streptococcus.
• Staphylococci and streptococci are nonmotile and do not form
spores.
• Both staphylococci and streptococci are gram-positive cocci,
but they are distinguished by two main criteria:
• (1) Microscopically, staphylococci appear in grapelike clusters,
whereas streptococci are in chains.
• (2) Biochemically, staphylococci produce catalase (i.e., they
degrade hydrogen peroxide), whereas streptococci do not.
STREPTOCOCCUS
 GENERAL CHARACTERISTICS OF
STREPTOCOCCI

•Gram-positive spherical/ovoid cocci

arranged in long chains OR pairs
•Non-spore-forming, nonmotile
•Facultative anaerobes
•Do not form catalase, but have a
peroxidase system so CATALASE
NEGATIVE .
 CLASSIFICATION:
• One of the most important criteria for identification is the type of
HEMOLYSIS.
• “HEMOLYSIS”
• DEFINATION: The destruction or dissolution of red blood cells,
with subsequent release of hemoglobin.
 CLASSIFICATION OF STREPTOCOCCI ON
THE BASIS OF TYPES OF HEMOLYSIS:
1:HEMOLYTIC STREPTOCOCCI:

ALPHA-HEMOLYTIC STREPTOCOCCI

BETA-HEMOLYTIC STREPTOCOCCI

2:NONHEMOLYTIC STREPTOCOCCI.(GAMMA)
1:ALPHA-
INCOMPLETE,
2:BETA-
COMPLETE,
3:GAMMA-NO
 BETA-HEMOLYTIC STREPTOCOCCUS:

Beta hemolytic
streptococci again
subdivided into
subgroups from
A,B,C,D-,-,-U known as
Lancefield classification
system on the basis of
difference in cell wall
antigen called C
carbohydrate.
 BETA HEMOLYTIC STREPTOCOCCI:

Group A: -hemolytic Streptococcus pyogenes

Group B: -hemolytic S. agalactiae
Group D: VARIABLE HEMOLYSIS
S. bovis (Non enterococci) , Enterococcus faecalis, E.
faecium (Enterococcus )

ALPHA HEMOLYTIC STREPTOCOCCI:

STREPTOCOCCUS PNEUMONIAE,
VIRIDANS STREPTOCOCCI (S.MITIS, S.SANGUIS,
S. MUTANS)
 Human Streptococcal Pathogens

•S. pyogenes
•S. agalactiae
•Viridans streptococci
•S. pneumoniae
•Enterococcus faecalis
 b-hemolytic S. pyogenes

• Most serious streptococcal pathogen

• Most frequent bacterial cause of pharyngitis

• Inhabits throat, nasopharynx, occasionally skin

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 PATHOGENESIS
• ANTIGENS PRESENT IN THE CELL ENVELOP

• INFLAMMATION-RELATED ENZYMES

• TOXINS AND HEMOLYSINS

 Virulence Factors of b-Hemolytic
S. Pyogenes (ANTIGENS)

Produces surface antigens:

• C-carbohydrates – protect against lysozyme

• M-protein –Important virulence factor and

determines the type of group A beta hemolytic
streptococci, contributes to resistance to
phagocytosis as it protrudes from the outer surface
of cell and interferes with ingestion by phagocytes.

18
 Virulence Factors of b-Hemolytic
S. Pyogenes

Extracellular toxins:
1. Streptolysins – hemolysins; streptolysin O (SLO)
and streptolysin S (SLS) – both cause cell and
tissue injury
2. Erythrogenic toxin – induces fever and typical red
rash
3. Pyrogenic Exotoxin A – strong monocyte and
lymphocyte stimulants; cause the release of tissue
necrotic factor (Similar to staphylococci toxic shock
syndrome toxin)
4. Exotoxin B: rapidly destroying tissue cause
necrotizing fasciilitis.

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 Virulence Factors of b-Hemolytic
S. Pyogenes

Extracellular enzymes
Streptokinase – (fibrinolysin) digests fibrin in clots ,
thrombi and emboli.

Hyaluronidase – breaks down connective tissue

which aids the spread of streptococci.

DNase – hydrolyzes DNA.

20
 TRANSMISSION:

• Most streptococci are part of normal flora but

produce disease when they gain access to
tissues or blood.
• Transmission – contact, droplets, food, fomites
• Portal of entry generally skin or pharynx
• Children predominant group affected for
cutaneous and throat infections
• Systemic infections and progressive sequelae
possible if untreated

21
 CLINICAL DISEASE

S. PYOGENES causes three types of diseases:

PYOGENIC DISEASES:
 a//-Pharyngitis
 b//-skin infections
TOXIGENIC DISEASES:
 a//-scarlet fever
 b//-toxic shock syndrome
IMMUNOLOGIC DISEASES:
 a//-rheumatic fever
 b//-acute glomerulonephritis

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PHARYNGITIS AND TONSILLITIS

23
 Streptococcal
skin infections

24
 Group B: Streptococcus
Agalactiae
• Regularly resides in human vagina,
pharynx, and large intestine
• Can be transferred to infant during delivery
and cause severe infection
• Most prevalent cause of neonatal
pneumonia, sepsis, and meningitis
• Pregnant women should be screened and
treated
• Wound and skin infections and endocarditis
in debilitated people
 Group D Enterococci
• Group D:
• Enterococcus faecalis, E. faecium, E.
durans
• Normal colonists of human large
intestine
• Cause opportunistic urinary, wound, and
skin infections, particularly in debilitated
persons.
 a-Hemolytic Streptococci:
Viridans Group

• Large complex group

• Streptococcus mutans, S. oralis, S.
salivarus, S. sanguis, S. milleri, S. mitis
• Most numerous and widespread residents of
the gums and teeth, oral cavity, and also
found in nasopharynx, genital tract, skin
• Not very invasive; dental or surgical
procedures facilitate entrance

27
 Viridians Group
• Bacteremia, meningitis, abdominal infection,
tooth abscesses cause mainly by this group.
• Most serious infection – subacute
endocarditis – Blood-borne bacteria settle
and grow on heart lining or valves
• Persons with preexisting heart disease are
at high risk
• S. mutans produce slime layers that adhere
to teeth, basis for plaque
• Involved in dental caries
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 Cellulitis

Cellulitis

is usually caused by bacteria –

specifically

1- group A streptococcus (also

known as Streptococcus
pyogenes),

2- Staphylococcus aureus
(including MRSA), and, in young
children,

3- Haemophilus influenzae type B.

 STAPHYLOCOCCUS

• Diseases:
• Staphylococcus aureus causes abscesses , various pyogenic
infections (e.g., endocarditis, septic arthritis, and osteomyelitis),
food poisoning, scalded skin syndrome and toxic shock
syndrome.
• It is one of the most common causes of
• 1- hospital-acquired pneumonia,
• 2- septicemia,
• 3- surgical-wound infections.
 STAPHYLOCOCCUS

• It is an important cause of skin and soft tissue infections, such

as folliculitis, cellulitis, and impetigo .

• It is the most common cause of bacterial conjunctivitis.

• Methicillin-resistant Staphylococcus aureus (MRSA) is the most
common cause of skin abscesses in the United States.
• It is also an important cause of pneumonia, necrotizing fasciitis,
and sepsis in immunocompetent patients.
 STAPHYLOCOCCUS SPECIES

• Staphylococcus epidermidis
• causes prosthetic valve endocarditis and prosthetic joint
infections.

• It is the most common cause of central nervous system shunt

infections and an important cause of sepsis in newborns.

• Staphylococcus saprophyticus
• causes urinary tract infections, especially cystitis.
• Kawasaki syndrome is a disease of unknown etiology that may
be caused by certain strains of S. aureus.
 Important Properties

• Staphylococci
• are spherical gram-positive cocci arranged in irregular grapelike
clusters.
• All staphylococci produce catalase, whereas no streptococci do
(catalase degrades H2O2 into O2 and H2O).

• Catalase
• is an important virulence factor. Bacteria that make catalase
can survive the killing effect of H2O2 within neutrophils
 Procedure of catalase test (Slide Test)

• 1- Transfer a small amount of bacterial colony to a surface of

clean, dry glass slide using a loop or sterile wooden stick

• 2- Place a drop of 3% H2O2 on to the slide and mix

• 3- A positive result is the rapid evolution of oxygen (within 5-10

sec.) as evidenced by bubbling
 Species:

• Three species of staphylococci are important human pathogens:

• S. aureus
• S. epidermidis
• S. saprophyticus
• Of these three, S. aureus is by far the most common and
causes the most serious infections.
•
 Species:

• Staphylococcus aureus is distinguished from the others

primarily by coagulase production.
• Coagulase is an enzyme that causes plasma to clot by
activating prothrombin to form thrombin.

• Thrombin then catalyzes the activation of fibrinogen to form the

fibrin clot
• Staphylococcus epidermidis and S. saprophyticus are often
referred to as coagulase-negative staphylococci
 Procedure: Slide Test
(to detect bound coagulase)
• 1- Place a drop of physiological saline on each end of a slide, or
on two separate slides.
• 2- With the loop, straight wire or wooden stick, emulsify a
portion of the isolated colony in each drops to make two thick
suspensions
• 3- Add a drop of human or rabbit plasma to one of the
suspensions, and mix gently.
• 4- Look for clumping of the organisms within 10 seconds.
• 5- No plasma is added to the second suspension to differentiate
any granular appearance of the organism from true coagulase
clumping.
 Tube Test (to detect free coagulase)
• 1- Dilute the plasma 1 in 10 in physiological saline ( mix 0.2 ml of
plasma with 1.8 ml of saline).
• 2- Take 3 small test tubes and label as T (Test), P (Positive
Control) and N (Negative Control). Test is 18-24 hour broth
culture, Positive control is 18-24 hr S. aureus broth culture and
Negative control is sterile broth.
• 3- Pipette 0.5 ml of the diluted plasma into each tube.
• 4- Add 5 drops (0.1 ml) of the Test organisms to the tube labelled
“T”, 5 drops of S. aureus culture to the tube labelled “P” and 5
drops of sterile broth to the tube labelled “N”
• 5- After mixing, incubate the three tubes at 35-37 Degree Celsius.
• Examine for clotting after 1 hours. If no clotting has occurred,
examine at 30 minutes intervals for up to 6 hours.
Result:
Coagulase Test.
 Why staph aureus colonies are golden?

• Staphylococcus aureus produces a carotenoid pigment called

staphyloxanthin which imparts a golden color to its colonies
 CONT:

• This pigment enhances the pathogenicity of the organism by

inactivating the microbicidal effect of superoxides and other
reactive oxygen species within neutrophils.

• Staphylococcus epidermidis:
Does not synthesize this pigment and produces white colonies.
The virulence of S. epidermidis is significantly less than that of S.
aureus.
Two other characteristics further distinguish these species,
namely, S. aureus usually
ferments mannitol and hemolysis red blood cells,
whereas S. epidermidis and S. saprophyticus do not
 Hemolysis of red cells WHY ?

• by Hemolysins produced by S. aureus is the source of iron

required for growth of the organism.
• The iron in hemoglobin is recovered by the bacteria and utilized
in the synthesis of cytochrome enzymes used to produce
energy.
• More than 90% of S. aureus strains contain plasmids that
encode a-lactamase, the enzyme that degrades many, but not
all, penicillin's.
• Some strains of S. aureus are resistant to the β-lactamase–
resistant penicillins, such as methicillin and nafcillin, by virtue of
changes in the penicillin-binding proteins (PBP) in their cell
membrane. Genes on the bacterial chromosome called mecA
genes encode these altered PBPs.
 CONT:

• These strains are commonly known as methicillin resistant S.

aureus (MRSA) or nafcillin-resistant S. aureus (NRSA).
• MRSA causes both health care-acquired (HCAMRSA) and
community-acquired (CA-MRSA) infections.
• MRSA currently accounts for more than 50% of S. aureus
strains isolated from hospital patients in the United States.
• CA-MRSA is a very common cause of community-acquired
staphylococcal infections.

• The most common strain of MRSA in the United States is the

“USA300” strain
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