Genus STAPHYLOCOCCUS
Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India
Introduction
Staphyloccocci - Greek “staphyle” (bunch of
grapes) and “Kokkos” = berry, meaning
bacteria occurring in grapelike clusters or
berry.
>40 species are known
Occur as commensal on skin and mucous
membrane
Natural habitat: Nostril and skin (Present on
the skin and mucus membrane)
Include major human pathogen
•Some act as opportunistic pathogens
causing pyogenic infections.
•Hardy organisms surviving many non-
physiologic conditions
History
Robert Koch (1878)- first to see staphylococci in pus
specimen
Louis Pasteur (1880)- first to cultivate in liquid
medium
Sir Alexander Ongston (1881)- named the bacteria as
“Staphylococcus”
Morphology & Biochemical Characteristics
Gram-positive cocci, 0.5-1.5 µm in diameter
Occur characteristically in group, also singly and in pairs
Form irregular grapelike clusters (since divide in 3 planes)
Non-motile
Non- spore forming
Catalase positive
Oxidase negative
Few strains are capsulated
Mostly aerobic or facultative anaerobic (02 species S. anaerobius
and S. saccharolyticus are anaerobic and catalase negative)
Grow in media containing 10% NaCl at temp 18 to 40
0
C
Coagulase positive S. aureus, S. intermedius, and coagulase
variable S. hyicus are important pathogens of domestic animals.
Coagulase negative staphylococci are low virulence, some
occasionally cause disease in humans and animals.
Classification
S. albus , S. aureus , S. citrus on Nutrient Agar
•Based on pigment production:
S. aureus : golden-yellow pigmented
colonies
S. albus : white colonies
S. citrus : lemon yellow colonies
•Based on pathogenecity:
Pathogenic: i.e. S. aureus
Opportunistic pathogens: S.
epidermidis, S. saprophyticus
Non-pathogenic: S. albus, S. citrus, S.
hominis etc.
•Based on coagulase production:
Coagulase positive: S. aureus
Coagulase negative: S. epidermidis, S.
saprophyticus
Usual Habitat
Staphylococci occur worldwide as commensals on skin of
animals and man.
Also found on mucous membrane of upper respiratory tract and
lower urogenital tract and as transients in digestive tract.
They are stable in the environment.
Some staphylococcal species exhibit affinity for particular
animal species.
Transfer of S. aureus between animals and man is limited.
Antigenic Structure
Antigenic structure
•Capsule
•Peptidoglycan
•Teichoic acid
•Protein A
Structure
CAPSULE
loose fitting polysaccharide layer
(slime layer)
protects bacteria by inhibiting
chemotaxis and phagocytosis
facilitates adherence of bacteria to
catheters and synthetic materials
PEPTIDOGLYCAN
half of the cell wall
consist of layers of glycan chains
with alternating subunits of N –
acetylmuramic acid and N-
acetylglucosamine
has endotoxin like activity
Structure.....
TEICHOIC ACID
Phosphate containing polymers bound
to peptidoglycan layer or to cytoplasmic
membrane - mediates the attachment of
staphylococcus to mucosal surfaces
S. aureus: Ribitol teichoic acid with N-
acetylglucosamine (Polysaccharide A)
S. epidermidis: Glycerol teichoic acid
with glucosyl residues
(polysaccharide B)
Structure.....
PROTEIN A
Covalently linked to peptidoglycan
Has affinity to Fc region of Immunoglobulins
Blocks opsonization and phagocytosis
Structure.....
COAGULASE
Bound coagulase or Clumping factor
binds fibrinogen, convert to insoluble
fibrin causing staphylococcus to clump
Other SURFACE PROTEINS
Collagen, Elastin, fibrinogen and
fibronectin binding proteins
CYTOPLASMIC MEMBRANE
Osmotic barrier for the cell and provides
an anchorage for the biosynthetic and
respiratory enzyme
MSCRAMMs
Microbial Surface Component Recognizing Adhesive Matrix
Molecules – include
Fibronectin-binding protein
Collagen-binding protein
Interacting with host cells
S. aureus colonizes the skin of mammals
S. aureus infections often begin at some breach in the
epithelial barrier
MSCRAMMs enhance the bacterial attachment to host cells
Virulence Factors in Staphylococci
Virulence Factors
Virulence Factors (contd....)
Virulence Factors (contd....)
Virulence Factors (....contd)
Toxins
A. 5 Cytolytic or membrane damaging toxins
1.Alpha hemolysin
2.Beta hemolysin
3.Gamma hemolysin
4.Delta hemolysin
5.Panton Valentine leucocidin
B. 2 Exfoliative toxins
C. Toxic Shock Syndrome Toxin (TSST-1)
D. >23 Enterotoxins
Cytotoxins
Alpha toxin –
disrupts the smooth muscle in blood vessels
toxic to erythrocytes, hepatocytes, platelets, cultivated cells
integrates to host cell membrane - pores - efflux of K and influx
of Na, Ca - osmotic swelling - cell lysis
septic shock
Beta Toxin
Sphingomyelinase C
Specific for sphingomyelin and lysophosphatidylcholine
Toxic to RBC, WBC, Macrophage and fibroblast
Catalyze hydrolysis of membrane phospholipids in susceptible
cells
Tissue destruction and abscess formation
Delta toxin
disrupts cell membrane
toxic to variety of cells
Lyse neutrophils - release of lysosomal enzymes - damage
surrounding tissues
Cytotoxins....
Gamma toxin and Panton Valentine leucocidin
Both damage membrane of susceptible cells
Lyse nuetrophils and macrophages
Cell lysis is mediated by pore formation
Cause necrotizing skin infection
PVL - potent leukotoxicity
Exfoliative toxin
•ETA - heat stable
•ETB – heat labile
•Serine protease
•Exposure - splitting of desmosomes or intercellular bridges in
the stratum granulosum epidermis and causes its separation
from underlying tissue, resulting in a blistering and exfoliating
disease of skin
•Common in neonates – ETA and ETB binds to GM4 like
glycolipids present in neonates
TSST-1
•Formerly pyrogenic exotoxin C and entertoxin F
•Induce cytokine release from macrophage and T lymphocytes
•heat & proteolysis resistant
•Superantigens
•Penetrate mucosal barrier
•Increase sensitivity to endotoxin
•Produce leakage of endothelial cells
•All S. aureus responsible for menstruation-associated TSS
produce TSST-1
•50 % of the strains responsible for other forms of TSS produce
TSST-1
Enterotoxins
•>23 types - till date
•Stable to heating, resistant
to hydrolysis
•Enterotoxin A – most
commonly associated with
disease
•Enterotoxin C and D-
contaminated milk products
•Enterotoxin B -
Pseudomembranous colitis
•Superantigens
Staphylococcal Enzymes
convert fibrinogen react with globulin
plasma factor to form
insoluble fibrin
staphylothrombin
Clumping factor
Cause formation of fibrin layer around abscess protecting
staphylococcus from phagocytosis
Coagulase
Bound Free
Staphylococcal enzymes
•Catalase: catalyze the conversion of toxic hydrogen peroxide to
water and oxygen
•Hyalurodinase: hydrolyzes hyaluronic acid in acellular matrix of
connective tissue - spread
•Fibrinolysin: Also k/a staphylokinase Dissolve fibrin clot- aid in
bacterial spreading
•Lipases: Lipases hydrolyse lipid to ensure survival in sebaceous
areas of the body
•DNase:
•Penicillinase: Plasmid mediated
•Fatty acid modifying enzyme (FAME): antibacterial lipid-
prolonged bacterial survival
Pathogenesis
•Staphylococci are pyogenic bacteria, they often cause suppurative
infections.
•Minor trauma or immunosuppression may predispose to development
of infection.
•Adhere to damaged skin, mucosa or tissue surfaces
–At these sites, they evade defence mechanisms of the host, colonize
and cause tissue damage
•Structural features including capsular polysacchrade, peptidoglycan,
teichoic acid and protein A interfere with opsonization.
•Cell wall proteins may bind to fibronectin, collagen, elastin and
fibrinogen may facilitate bacterial attachment to tissues.
•Production of coagulase is important virulence factor.
•Other virulence factors including enzymes also play important role.
•S. aureus produces disease by
–Multiplying in tissues
–Liberating toxins,
–Stimulating inflammation
Predisposing Factors Leading to S. aureus Infections
Skin damage: burns, cuts, sutures
Reduced Chemotaxis: burns, diabetes, cancer
Reduced Phagocytosis: diabetes, complement deficiency,
immunoglobulin deficiency, genetic defect in phagocytes
Age: very young or very old
Clinical Infections: in Animals
Since staphylococci occur as both commensal of skin and mucous
membrane and as environmental contaminants, infections can be
either endogenous or exogenous in origin.
Many infections are opportunistic and associated with trauma,
immunosuppression, inter-current parasitic or fungal infections,
allergic conditions or endocrine or metabolic disturbances.
COAGULASE POSITIVE STAPHYLOCOCCI ARE RESPONSIBLE FOR
MOST OF THE INFECTIONS.
COAGULASE NEGATIVE STAPHYLOCOCCI ARE ALSO CAPABLE OF
CAUSING DISEASE IN ANIMALS.
Important staphylococcal diseases include mastitis, tick pyaemia,
exudative dermatitis, botryomycosis and pyoderma.
Diseases in Animals by Coagulase positive Staphylococci
Species HostsClinical Conditions
Staphylococcus
aureus
CattleMastitis,
Udder Impetigo
SheepMastitis,
Tick Pyaemia (Lambs),
Dermatitis,
Benign Folliculitis (Lambs)
Goat Mastitis,
Dermatitis
Pigs Botryomycosis of mammary glands,
Impetigo of mammary glands
HorsesBotryomycosis of spermatic cord (Scirrhous cord),
Mastitis
Dogs,
Cats
Suppurative conditions similar to those caused by S. intermedius
PoultryBumblefoot,
Omphalitis in chicks,
Arthritis and Septicaemia in turkeys
Species Hosts Clinical Conditions
Staphylococcus intermediusDogs, Pyometra,
endometritis,
cystitis,
otitis externa and
other suppurative conditions
Cats Varous pyogenic infections
CattleMastitis (Rare)
S. hyicus (50% are CoPS) Pigs Exudative dermatitis (Greasy Pig disease)
CattleMastitis (Rare)
S. aureus ssp anaerobius Sheep Lymphadenitis
S. delphini DolphinsSuppurative skin lesions
S. Schleferi
S. schleiferi ssp. coagulans (cf. S.
schleiferi ssp. schleiferi is CN)
Dogs Otitis externa
S. pseudintermedius
S. lutrae
Diseases in Animals by Coagulase positive Staphylococci
Staphylococcal mastitis
Staphylococcal mastitis, mostly caused by S.
aureus, is a common form of bovine mastitis,
worldwide.
It may be subclinical, acute or chronic.
The majority of infections are subclinical.
Peracute and gangrenous forms are associated
with severe systemic reactions and can be life
threatening.
In gangrenous mastitis, the affected quarter,
which becomes cold and blue black, eventually
sloughs.
Tissue necrosis is attributed to alpha-toxin which
causes contraction and necrosis of smooth
muscles in blood vessel walls, impeding blood
flow in the affected quarter. In addition, this
toxin causes release of lysosomal enzymes from
leucocytes.
Tick Pyaemia
Tick pyaemia is an infection of lambs by S. aureus.
Infection is confined to hill grazing regions where
there are suitable habitats for the ticks Ixodes
ricinus.
Ixodes ricinus is a vector for the Rickettsial agent
of tick-borne fever, Ehrlichia phagocytophila, which
can cause immunosuppression in lambs and may
predispose to staphylococcal infection.
Lambs carry S. aureus on their skin and nasal
mucosa and infection occurs through minor skin
trauma including tick bites.
Tick pyaemia is characterized either by septicemia
and rapid death or by localized abscess formation
in many organs.
Clinical manifestations include arthritis, posterior
paresis and ill thrift.
The condition can be of considerable economic
importance on some farms where up to 30% of
lambs between 2 and 10 weeks of age can be
affected in spring and early summer.
Exudative dermatitis (Greasy Pig Disease)
This disease is caused by S. hyicus.
Occurs worldwide in sucklers and weaned pigs
up to 3 months of age.
It is highly contagious and characterized by
widespread excessive sebaceous secretion,
exfoliation and exudation on the skin surface.
Affected pigs, which are anorexic, depressed
and febrile, have an extensive, non-pruritic
dermatitis with greasy exudate.
Piglets under 03 weeks of age may die within 24
to 48 hours.
Morbidity range from 20-100%, and morality
rates can reach 90% in severely affected litters.
S. hyicus can be isolated from the vaginal
mucosa and skin of healthy sows.
The organisms probably enters the skin of
young pigs through minor abrasions such as bite
wounds.
Predisposing stress factors include agalactia in
the sow, intercurrent infections and weaning
stress.
Botryomycosis
Botryomycosis; also known as bacterial pseudomycosis is a rare
chronic suppurative granulomatous bacterial infection that
affects the skin, and sometimes the viscera, often caused by S.
aureus.
Botryomycosis has been known to affect humans, horses, cattle,
swine, dogs and cats.
It can occur within a few weeks of castration in the horse due to
the infection of the stump of the spermatic cord (scirrhous
cord).
Botryomycosis can also occur in mammary tissues of the sows.
The lesion is composed of a mass of fibrous tissue containing
foci of pus and sinus tracts.
The disease was originally discovered by Otto Bollinger (1843–
1909) in 1870, and its name was coined by Sebastiano Rivolta
(1832–1893) in 1884.
The name refers to its grape-like granules (Gr. botryo = grapes)
and the mistakenly implied fungal etiology (Gr. mykes = fungus).
In 1919 the bacterial origin of the infection was discovered.
Bumblefoot in Poultry
Bumblefoot is an infection caused by the Staphylococcus which
enters the chicken’s system through a cut, scratch, injury.
The infection creates an abscess full of pus.
It affects all species of poultry and occurs worldwide.
Staphylococcal infections of dogs and cats
Staphylococcus intermedius is commonly isolated from
pyometra, otitis externa and other suppurative conditions
including mastitis, endometritis, cystitis, osteomyelitis and
wound infections.
Occasionally, similar suppurative conditions are caused by S.
aureus.
DISEASES – In human
•Due to direct effect of
organism
–Local lesions of skin
–Deep abscesses
–Systemic infections
•Toxin mediated
–Food poisoning
–Toxic shock syndrome
–Scalded skin syndrome
1) Cutaneous Infections
•Folliculitis: It is inflammation of the hair follicles.
•A small red bump or pimple develops at infection sites of hair follicle.
•Sty: A sty is folliculitis affecting one or more hair follicles on the edge of the
upper or lower eyelid.
Cutaneous Infections (contd....)
Furuncle/boils:
•Furuncle is deep seated infection,
originating from folliculitis, (if
infection extends from follicle to
neighbouring tissue)
•Causes redness, swelling, severe pain
•Commonly found on the neck, armpit
and groin regions
Carbuncle:
•Carbuncle is an aggregation of
infected furuncles.
•Carbuncles may form large abscesses.
•It is a large area of redness, swelling
and pain, punctuated by several sites
of drainage pus.
•
Cutaneous Infections(contd....)
Impetigo:
•a very superficial skin infection common in children, usually
produces blisters or sores on the face, neck, hands, and diaper
area.
•It is characterized by watery bristles, which become pustules
and then honey coloured crust
impetigo with vesicles, pustules, and sharply
demarcated regions of honey-colored crusts.
2) Deep Infections
Osteomyelitis: Inflammation of bone
•Bacteria can get to the bone
–Via bloodstream
–Following an injury
Clinical features: pain, swelling,
deformity, defective healing, in
some case pus flow,
Diagnosis: X-ray, MRI, bone aspirates
Deep Infections (contd....)
Periostitis: inflammation of
periosteum
•Clinical features: fever, localised
pain, leucocytosis
•Diagnosis: needle aspiration of
subperiosteal fluid
Deep Infections(....contd)
•Endocarditis: It is
an inflammation of
the inner layer of
the heart, the
endocardium
•Endocarditis
occurs when
bacteria enter
bloodstream,
travel to heart, and
lodge on abnormal
heart valves or
damaged heart
tissue.
3) Exfoliative Disease
•Exfoliate - scaling off tissues in layers
•It is a skin disease in which outer layer of
epidermis gets separated from the
underlying tissues.
•Exfoliative toxin produced by S. aureus is
responsible for this.
•Also known as ‘Staphylococcal skin scalded
syndrome’ SSSS
•Previously called dermatitis exfoliativa,
pemphigus neonatorum, Lyell’s disease and
Ritter’s disease
•Epidermal toxin produced by S. aureus at skin
and is carried by bloodstream to epidermis,
where it causes a split in a cellular layer i.e.,
this toxin separates outer layer of epidermis
from underlying tissue
splitting of desmosomes or intercellular bridges in the
stratum granulosum epidermis and causes its separation
from underlying tissue, resulting in a blistering and
exfoliating disease of skin
Types of SSSS:
Severe form Milder form
In new born - Ritter’s disease - Pemphigus
neonatorum
In older patients - Toxic epidermal - Bullous
necrolysis impetigo
Toxic epidermal necrolysis
Ritter’s disease
Bullous impetigo
Pemphigus neonatorum
4) Toxic Shock Syndrome
•Caused when Toxin shock syndrome toxin (TSST) liberated by S.
aureus enters bloodstream
•It is fatal multisystem disease presenting with fever,
hypotension, myalgia, vomiting, diarrhoea, mucosal hyperemia
and erythematous rash which desquamates subsequently.
5) Staphylococcal Food Poisoning
•Caused when consuming food in which S. aureus
has multiplied and formed enterotoxins.
•It usually occurs when preformed toxin is
ingested with contaminated food.
•Enterotoxin is responsible for manifestations of
staphylococcal food poisoning.
•> 23 types of enterotoxin are currently known,
named A-E and G- w.
•The toxin acts directly on the autonomic nervous
system to cause the illness, rather than gut
mucosa
•Symptoms: Nausea, Vomiting, Severe abdominal
cramp, Diarrhoea, Sweating, Headache etc.
•The common food items responsible are - milk
and milk products, meat, fish and ice cream.
•Source of infection- food handler who is a carrier.
•Incubation period- 2 to 6 hours.
•The illness is usually self limited, with recovery in
a day or so.
Laboratory Diagnosis
A. Bacteriological Investigation:
•Specimens:
–Pus: from wound or abscess
or burns
–Nasal Swab: from suspected
carrier
–Food: to diagnose
staphylococcal intoxication
–Blood: to diagnose
endocarditis and bacteremia
–Sputum: to diagnose lower
respiratory tract infection
–Milk: To detect mastitis
STAPHYLOCOCCI
Gram’s Stain:
•These are spherical cocci.
•Approximately 1 μm in
diameter.
•Arranged
characteristically in grape
like clusters.
•They are non motile and
non sporing.
•A few strains possess
capsules.
CULTURE
Media used:
i) Non selective media: Nutrient agar,
Blood agar,
MacConkey’s agar.
ii) Selective media: Mannitol salt agar
Baired Parker agar
Ludlam’s medium
Salt-milk agar,
Cultural Characteristics
I.On nutrient agar- The colonies are large, circular, convex,
smooth, shiny, opaque and easily emulsifiable. Most strains
produce golden yellow pigments.
II.On MacConkey’s agar- The colonies are small & pink in colour.
III.On blood agar- Most strains produce β- haemolytic colonies.
Double zone of hemolysis
Outer zone: incomplete
hemolysis (Alpha)
Inner zone: complete hemolysis
(beta-hemolysis)
Double zone hemolysis by S.
aureus. S. intermedius and S.
pseudintermdedius also produce
double-zone hemolysis
Laboratory Diagnosis (contd....)
•Culture and isolation:
–Specimens are cultured on BA plate and are
incubated @ 37 °C for 24 hours
–After incubation, BA plate is observed for
significant bacterial growth (> 2mm in
diameter)
–Then, Gram-staining is performed of the
isolated organisms
–Then, sub-cultured on NA plate for further
biochemical tests
•Tube coagulase test:
–i. Mix 0.5ml of human plasma with 0.1 ml of
an overnight broth culture of S. aureus
–ii. Incubate the mix in a water bath @ 37°C for
3-6 hours
–Result: plasma clots and doesn’t flow if the
tube is inverted
Culture
•Aerobes and facultative anaerobes
•Optimum temp. for growth= 37°C
•Optimum pH for growth= 7.4-7.5
•On Nutrient agar,
–golden yellow and opaque colonies with smooth
glistening surface, 1-2 mm in diameter (max.
pigment production@22 °C)
•On Blood agar,
–golden yellow colonies, surrounded by a clear zone
of hemolysis (beta-hemolysis), esp. when incubated
in sheep or rabbit blood agar in atmosphere of 20%
CO2
•On MacConkey agar,
–Smaller colonies than those on NA (0.1-0.5 mm) and
are pink coloured due to lactose fermentation
•On Mannitol salt agar,
–S. aureus ferments mannitol and appear as yellow
colonies
–MSA is a useful selective medium for recovering S.
aureus from faecal specimens, when investigating
food poisoning
Biochemical Properties
•Catalase test: positive
•Oxidase test- negative
•Coagulase test= positive
•Sugar Fermentation tests:
Ferment glucose, lactose,
maltose, sucrose and mannitol,
with production of acid but no gas
Mannitol fermentation carries
diagnosis significance
•Indole test= negative
•MR test= positive
•VP test= positive
•Urease test= positive
•Hydrolyse gelatin
•Reduces nitrate to nitrite
•Phospahatase= positive
•DNase test= positive
•Gelatin liquefaction test- Positive.
•Produces Lipase
•Produces Phosphatase
•Produces Thermostable nuclease.
Differentiation of Staphylococcus species
Species Colony
Colour
Hemolysis
on sheep
blood ager
Tusbe
coagulase
test
Slide
Coagulase
test
Acetoin
Producti
on
Maltose
utilizatio
n
S. aureusGolden
yellow
+ + + + +
S.
intermedius
White + + V - +
S. hyicusWhite V V - - -
S. aureus ssp
anaerobius
White + + - - NA
S. DelphiniWhite + + - - NA
S. SchleferiWhite + + - + NA
Species Catalase Coagulase Mannitol Novobiocin
S. aureus positive positive positive sensitive
S. epidermidis positive negative negative sensitive
S. saphrophyticus positive negative negative resistant
Properties of Staphylococcus species
Differentiation of Gram Positivi Cocci
Organism Gram
Staining
Catalase
Production
Coagulase
Production
OxidaseO-F
test
Bacitracin
Sensitivity
(0.04U)
StaphylococciIrregular
clusters
+ + - F R
Streptococci
and
Enterococci
Chains - - - F R
Micrococci Packets of
four
+ - + O S
Treatment
•Antibiotic susceptibility testing should be done before
treatment.
•Drug resistance is common.
•Benzyl penicillin is the most effective antibiotic, if the strain is
sensitive.
•Cloxacillin or Methicillin is used against beta-lactamase
producing strains.
•Methicillin Resistant Staphylococcus aureus (MRSA) strains
have become common, now.
•Vancomycin is used in treatment of infections with MRSA
strains.
•Vancomycin resistance is very rare – so far
•MRSA were considered to be Hospital-acquired
•But, Community-acquired cases now (CA MRSA)
•Methicillin-resistant S. epidermidis - MRSE
•VRSA= vancomycin resistant Staphylococcus aureus
• VISA= vancomycin intermediate Saphylococcus aureus
Prevention
Schematic Diagram for Identifying Staphylococcal Species
Summary Micrococcaceae
Staph. aureus Staph.
Epidermidis
Staph.
saprophyticus
Micrococcus
Colony
Morphology
Opaque,
smooth, raised,
entire, white-
golden(cream)
Opaque,
smooth, raised,
entire, gray-
white
Opaque,
smooth, raised,
entire,
butyrous,
glossy, white-
yellow
Opaque,
smooth, raised,
white, bright
yellow
Hemolysis Most are beta
hemolytic
Non-hemolytic Non-hemolytic Non-hemolytic
Gram
morphology
GPC in
clusters, pairs,
short chains or
singly
GPC in
clusters, pairs,
short chains or
singly
GPC in
clusters, pairs,
short chains or
singly
GPC in pairs
and tetrads
Catalase Pos Pos Pos Pos
Glucose
fermentation
Fermenter Fermenter Fermenter Oxidizer
Modified
Oxidase
Neg Neg Neg Pos
Bacitracin
susceptibility
(Taxo A
0.04U)
Resistant Resistant Resistant Sensitive
Coagulase
Production
(tube)
Pos Neg Neg N/A
Clumping
factor (slide or
latex
Coagulase test)
Pos Neg Neg Neg
Thanks
Acknowledgement: All the material/presentations available online on the subject
are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the source and
authenticity of the content.
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