Gram Positive Cocci

 Staphylococci and Streptococci - Medically

important Gram positive cocci.

They are differentiated

 Biochemically, by the presence or absence of
catalase activity, and
 Microscopically, by their arrangement; whether
cluster or chains
 Macroscopically, by colony characteristic- pin head
or pin point colony with color and on hemolysis.
Staphylococcus
Introduction
General feature
Classification
 Staphylococcus
 “Staphylococcus” comes from– Greek word
Staphyle- means a bunch of grapes
  Responsible for more than 80% of pyogenic
infection
  2nd only to E.coli as a cause of Nosocomial
infection

GENERAL FEATURES:
 Gram positive cocci, arranged in irregular grape like
clusters
 Usually Non-capsulated, Non-motile, non-spore
forming, catalase and coagulase positive,
 Facultative anaerobes
 Classification
S t a p h y lo c o c c o u s

A e r o b ic A n a e r o b ic

C o a g u la s e p o s it iv e C o a g u la s e n e g a t iv e P e p to c o c c o u s

S .a u re u s S . e p id e r m id is S . s a p r o p h y t ic u s
 Cultural Characteristics
 Incubation temperature - 370C, but can grow in a
wide range
Routine culture
 Grow in ordinary media, such as nutrient agar,
nutrient broth
 Blood agar media is commonly used

 Beta hemolytic but zone of hemolysis is narrower

 Pin head colony
 Produce opaque, smooth, low convex, glistening
colonies
 Cultural Characteristics
Selective media
 Media containing 5-10% NaCl, such as
 Nutrient agar with 7-10% NaCl

 Mannitol salt agar – Selective for Staph. aureus

 Source of infection and
Transmission
Healthy Carriers
 Normal component of human flora
 Grows harmlessly on the anterior nare and
perineum of 20-40% of healthy individual
 Carriers serve as a source of infection to
themselves and others
 Spread by the hands, handkerchiefs, clothing

Important cause of nosocomial infection

Animals
 Milk from a cow with mastitis causing
staphylococcal food poisoning
 Virulence Factors
Surface Factors
 Essential components for colonization and
evasion of host defense
Surface factors comprises
 Cell wall - Peptidoglycan, Teichoic acid
 Cell surface proteins – Protein A,
Polysaccharide Capsules
 Peptidoglycan (PG)

• Elicits IL 1(endogenous pyorogen)

• Has endotoxin like activities and causes
endotoxic shock
• Can activate complement
 Teichoic Acid (TA)
 Composed of ribitol phosphate
 Mediate attachment through binding to
Fibronectin
 Antigenic and anti-TA Abs are detected in
endocarditis due to S. aureus
 Protein A
 Specialized structure coating the surface
 It has a unique affinity to bind to the Fc
receptor of IgG instead of FAb
 Thus prevents antibody mediated immune
clearance
 It is anti-complementary and anti-
phagocytic
 Used in Co-agglutination reaction
 Staphylococcus
Immunoglobulin
(Fc can be attached to Protein A)

Protein A
(receptor for
immunoglobulin)

antigen

Co agglutination reaction

Use: Detection of Pneumococcal or other bacterial

Antigens In CSF (meningitis)
 Polysaccharide Capsules
 Outermost layer of some S. aureus strains

 Act as virulent factors by inhibiting phagocytosis

 Protect the organism from the complement

mediated attack

 Encapsulated staphylococci are able to spread

rapidly through tissue
 Staphylococcal Enzymes
Coagulase
S. aureus produces free and bound coagulase
Presence of coagulase differentiate S. aureus
from other staphylococci
Coagulase
 Causes oxalated or citrated plasma to clot

 It activates prothrombin to form thrombin, which

catalyzes fibrinogen to form fibrin clot
 Fibrin deposited on staphylococcal surface and
protect from phagocytosis
 Staphylococcal Enzymes
Coagulase
Detected by Tube method
 0.5 ml citrated plasma is taken in a
test tube
 A drop of fresh young culture is added

 Incubated at 370C for 1 to 4 hours

 Production of distinct clot indicate
positive result
Coagulase test
 Catalase

• Converts H2O2 into O2 and H2O.

• Staphylococcus aureus is catalase positive

H2O2

Procedure: =
H2 O
+O2

A staphylococcus colony is picked with a glass rod and dipped

into a test tube containing H2O2.
Formation of bubble indicates positive reaction.
 Hyaluronidase, Lipase and
Staphylokinase
 Hyaluronidase hydrolyzes
hyaluronic acid in
connective tissue and
facilitate spread of infection
 Lipase helps in colonization
in sebaceous areas,
essential for invasion in
cutaneous and
subcutaneous tissues
 Staphylokinase or
fibrinolysin have fibrinolytic
activity
 Nucleases and
other enzymes
Nucleases
 Heat resistant nucleases cleave nucleic acid
 Heating at 650C causes structural disruption
 But the changes are rapid and completely reversible

Other staphylococcal enzymes are

 Proteinase
 Phosphatase
 Penicillinase etc
Staphylococcal Toxins
Two types of toxins
Cytolytic Exotoxins
Superantigen Exotoxins
 Cytolytic Exotoxins

 Hemolysins
 Four distinct hemolytic toxins – α, β, γ and δ
 Among them α-toxin is best studied
 Leucocydin
 Super-antigen Exotoxins
 Enterotoxin
 Toxic Shock Syndrome Toxin and
 Exfoliatin Toxins
 Super antigen

= Nonspecific proliferation
of lymphocytes
T cell

= release of IL1, TNF

β α TCR
Class II MHC
α β
Super antigen

APC
 Super-antigen Exotoxins
Enterotoxin

 About 1/3rd of all S. aureus produce enterotoxin that

cause diarrhea and vomiting

 Major cause of food poisoning by preformed toxin

 8 types of enterotoxins isolated (A-E, G- I),

enterotoxin A is most frequently isolated

 Heat stable (1000C for 30 min) and resistant to

gastric and jejunal enzymes
 Super-antigen Exotoxins
Toxic Shock Syndrome Toxin-1
 Toxic shock syndrome toxin (TSST) – Cause Toxic shock
syndrome in tampon using menstruating lady and also in
nasal pack, as 5% lady contain S. aureus in vagina.

 TSST-1 – super antigen that stimulate release of

cytokines,

 Associated with fever, rash, shock, multi-system

dysfunction and desquamation of skin
 Exfoliative Toxins
(Epidermolytic Toxins)

 Cause staphylococcus scalded skin syndrome (SSSS)

 Toxin cause lyses of the intercellular attachment

between the cells of the granular layer of epidermis

 Loss of nail, hair, erythematous large bullae which

burst , produce ulcer. Loss of serous fluid leading to
electrolyte imbalance. Recovery within 7-10 days.

 Do not elicit an inflammatory response

 Pathogenesis and
Clinical Manifestation
Two types of syndrome

Pyogenic infections -through

direct invasion and tissue destruction

Intoxications -by producing toxin

Invasive Pyogenic
Infection
Two types of inflammatory response
Local infection – superficial and
deep
Systemic infection
Superficial localized infection
 Folliculitis – small, superficial abscesses
involving hair follicles, e.g., common sty

 Furuncles – an extension of folliculitis – large,

painful, raised nodules with focal suppuration

 Carbuncle – similar to furuncle but has

multiple foci and extends to deeper tissue;
systemic spread via bacteremia to other
tissue
Superficial localized infection
 Impetigo – localized, superficial, spreading
crusty skin lesion generally seen in children.
Also caused by Streptococcus pyogenes

 Wound infection – occur in patients after a

trauma or after a surgical procedure.
Organisms colonizing the skin are introduced
into the wound
Impetigo-Cutaneous abscess
Staph. Infection-MRSA
infection
MRSA infection
Staph infection
Staph infection
 Deep localized infection
 Osteomyelitis – S. aureus is the most
common cause of acute chronic infection of
bone
 Arthritis – common cause of septic arthritis in
children. Medical emergency, as pus can
rapidly cause irreparable damage to
cartilage. Also occur in adults receiving intra-
articular injections or have mechanically
abnormal joints.
 Systemic infections
 Acute endocarditis – may occur on normal or
prosthetic heart valves, especially right sided
endocarditis in intravenous drug users
 Septicemia – generalized infection with
sepsis or bacteremia that may originate from
any localized lesion
 Nosocomial infection – one of the most
common cause of hospital-acquired
infections, often of wound or bacteremia
associated with catheters
 Systemic infections
Pneumonia

S. aureus enter to the lung by two routs

 Aspiration of upper respiratory flora – primarily in
very young, elderly, and patients with cystic fibrosis,
influenza, COPD etc
 Hematogenous spread from a distant site – common
for patient with bacteremia or endocarditis

10% patient with pneumonia develop empyema

S. aureus is the most common cause of
nosocomial pneumonia
Staphylococcal food poisoning
 Occur within 2 to 6 hour of ingestion of contaminated
food with preformed toxin

 Symptoms start abruptly with nausea, vomiting,

crampy abdominal pain and diarrhea

 Self limited and resolve between 8 to 24 hours

 Foods tend to be protein-rich (pastry, ice cream,

salted meat), improperly refrigerated

 Gastro-enteritis triggered by local action of toxins on

G I Tract
Staphylococcal food poisoning
Enterotoxin induce Vomiting by
 When enterotoxin irritate the emetic receptor
in abdominal viscera
 Sensory stimulus reaches the vomiting centre
through vagus and sympathetic nerve

Enterotoxin induce diarrhea by

 Inhibition of water absorption by the lumen &

 Increased trans-mucosal fluid flux

Scalded skin syndrome (SSS)
 SSS primarily afflicts neonates and children

 Syndrome involves appearance of superficial

bullae

 Toxin attacks the intercellular adhesive layer of

the stratum granulosum of epidermis
 This cause marked epithelial desquamation
 Leads to separation of the epidermis at the
granular cell layer
 Coagulase negative
Staphylococci (CoNS)
 Major cause of nosocomial infection

 Most frequently isolated from blood of

hospitalized patients

 Most abundant and important CoNS recovered as

normal skin flora is S. epidermidis

 The second most important CoNS is S.

saprophyticus
Staphylococcus saprophyticus
 Second to E. coli as a cause of UTI among sexually
active women- named Honeymoon cystitis

 Most women with this infection have had sexual

intercourse within previous 24 hours

 S. saprophyticus can be distinguished from S.

epidermidis by its natural resistance to novobiocin

 Infection is readily amenable to therapy with

antibiotics commonly used to treat UTI
 Staphylococcus epidermidis
 Present in large numbers as part of the normal flora of
the skin

 Despite its low virulence, it is a common cause of

infection of intravenous catheter and prosthetic implants

 Major cause of sepsis in neonates and peritonitis in

peritoneal dialysis patients
 Laboratory Diagnosis
Identification depends largely on
 Microscopy
 Colony morphology
 Catalase positivity

Meticulous care must be taken during

specimen collection as they are widely
distributed in nature
 Specimens
 Pus from abscess
 Sputum from cases of pneumonia
 Surface swabs from wound
 Blood from cases of bacteremia, e.g., septic
shock, osteomyelitis, endocarditis
 CSF from suspected meningitis or brain abscess
 Feces, vomit or left over food - food poisoning
 Urine from UTI
 Anterior nasal or perineal swab from suspected
carriers
 Microscopy
 Gram positive cocci in seen as stained smear

Arrange in
 Grape-like clusters in agar media, and
 Single cell or small groups in

clinical specimens
 Culture

 Grow within 24h in nutritionally enriched media supplemented

with sheep blood

 Golden-yellow pigment with hemolysis (due to α-toxin) – in

blood agar

 S. aureus but not other staphylococci ferment mannitol

 So, selective and indicator media for S. aureus is mannitol

salt agar

 In mannitol salt agar- produce colonies surrounded by

yellow halo in 7-10% salt
mannitol salt agar: pH
indicator turns the agar
yellow in the presence of
a salt tolerant organism
 Identification and
differentiation
 Catalase tests – differentiate S. aureus from
Streptococcus

 Coagulase test – differentiate S. aureus from

CoNS (S. epidermidis and S. saprophyticus)

 Novobiocin sensitivity test – differentiate S.

epidermidis from S. saprophyticus
 MRSA, NRSA
 >90% S. aureus are resistant to penicillin G
 Treated with β-lactamase resistant penicillin

 ~20% S. aureus and 75% S. epidermidis are resistant

to methicillin, oxacillin and nafcillin due to change in its
penicillin binding protein in cell membrane.
 These MRSA, ORSA and NRSA are treated with
vancomycin, which also develop resistance in 2003.

 Vancomycin resistant strain are treated with Synercid,

linezolid.
 β-lactam drug plus β-lactamase inhibitor, clavulanic
acid also used.
Thank You