GRAM POSITIVE

COCCI

STAPHYLOCOCCUS
& STREPTOCOCCUS
 Description of microorganism

Classification Clinical findings

Morphology Diagnostic laboratory
Culture tests
Antigens Immunity
Toxins Treatment
Enzymes Prevention and control
Habitat & Transmission
Pathogenesis
 STAPHYLOCOCCUS AUREUS

 Spherical (cocci) with

0.8-1µm in diameter

Arranged :
 grape like cluster
 Gram positive,
 non motile,
 non-sporing & usually non
capsulated
 Aerobes and facultative
anaerobes
Cultural Characteristics
Nutrient agar

Colonies :
 large, circular,
 smooth white / golden yellow in colour

Pigmentation :
 Lipoprotein with carotene
- 22ºC &
- milk
- glycerol monoacetate
(incorporated in the medium)
 On slope -‘Oil paint’ appearance
golden yellow in colour
Cultural Characteristics

Blood agar :-
 β hemolytic colonies

 On sheep blood agar

 MSA
SELECTIVE MEDIA

Selective media:
 Salt-milk agar

 (8-10% NaCl),

 Salt agar,

 Ludlam’s medium
(lithium chloride & tellurite)

 Mannitol salt agar etc.

 Biochemical reactions

 Catalase &
coagulase positive

 Potassium tellurite
reduction – black
colonies

 Liquefy gelatin

coagulase catalase
Biochemical reactions

 Produce phosphatase

 Ferment sugars,
producing acid & no gas

 Reduces nitrates to nitrites,

 Both MR & VP positive,

 Fermentation of mannitol
 Virulence factors

Cell wall associated:

 Capsule

 Peptidoglycan

 Teichoic acid

 Protein A

 Clumping factor
Antigenic Structure

Teichoic acid:
 Major antigenic determinant

 Adherence to mucosal surface

 Protects against – complement mediated

opsonisation

 Absent in Staph. epidermidis

Extra cellular enzymes:

 Coagulase

 Staphylokinase,

 Hyaluronidase,

 Deoxyribonuclease,

 Lipase

 Phospholipase

 Protease
 Extracellular enzymes
Coagulase:

 An enzyme secreted into the medium

 Activates - Coagulase Reacting Factor (CRF) present in the
plasma,
 converts fibrinogen to fibrin clots the plasma.

 8 antigenic types : A-H, most strains producesA

 prevents opsonisation ,phagocytosis.

 Detected by TUBE COAGULASE TEST.

 Coagulase test

 The coagulase test differentiates [Link]

from other staphylococci
 Fibrogen coagulase Fibrin (visible clot)
 Slide test: detect bound coagulase
(clumping factor)
 Tube test: detect free coagulase, incubate
4 hours
 Continues….

Clumping factor (bound coagulase):

 Surface component
 causes clumping of organism when mixed with plasma

 Acts directly with fibrinogen in plasma

 Heat stable
 CLASSIFICATION
Depending on enzyme COAGULASE

 Coagulase positive-
Staphylococcus aureus

 Coagulase negative-
Staphylococcus epidermidis
Staphylococcus saprophyticus
[Link], [Link], [Link]
Other GPC-Micrococcus species
DNA test

A. Strongly positive
B. Weakly positive
C. Negative
 Mannitol fermentation

Negative (St epidermidis) Positive (St aureus)

Nitrate reduction test

Negative Positive
 Indole test

Negative
(St aureus)
Positive
Pathogenicity
 Pathogenicity

 30-50% of adults - in anterior nares , carry [Link]

perineum, axillae vagina
 Mode of transmission –contact/ fomites /droplets
 Entry - through damaged skin,
mucous membranes,
viral infections (esp. LRTI).
 Diseases of Staph. aureus –
Toxin mediated & Invasion.
 Toxin mediated: Exfoliative disease

 Epidermolytic toxin

 Seperates outer layer of epidermis from

underlying tissue
- blister formation
 Staphylococcus Scalded Skin Syndrome /
Ritter’s disease –newborns
 Exfoliative /toxic epidermal necrolysis-
older
 Milder-
pemphigus neonatorum, bullous impetigo
 Food poisoning
Enterotoxin

 Heat stable toxin -100ºC for 10 to 40 min.

 Nausea, vomiting and diarrhoea occurs

 IP-2 to 6 hours (preformed toxin).

Source:
 Cooked meat, fish, milk & milk products are mostly
responsible.
Toxin acts on: autonomic nervous system,
 antigenic neutralised by antitoxin
 types: 8 antigenic types, Type A –most common
 Detected: latex agglutination, ELISA
 Toxic Shock Syndrome Toxin-I

 Multisystem disorder with fever, hypotension,

myalgia, rash, vomiting, diarrhoea etc.

 Associated with colonisation of mucosal

surface by TSST producing strain belonging
to phage group I

 TSST-1 antibody- protective

 SUPER ANTIGENS

 TSST I & Enterotoxins - SUPER ANTIGENS

 Potent activators of T lymphocytes
 Stimulate T cells-without relation to epitope specificity
 An excessive & dysregulation of immune response

Release :
 cytokines,

 interleukins 1,2,

 tumour necrosis factor,

 interferon gamma
 Spectrum of diseases

S. aureusandDisease  Skin and soft tissue infections

 Musculoskeletal infections

Boils
Endocarditis FoodPoisoning
 Respiratory tract infections
Central nervous system
infections
Scalded Skin Syndrome Impetigo
Cellulitis

 Blood stream infections

ToxicShockSyndrome Ostermylelitis
Endophthalmitis Pneumonia
 Urinary tract infections
Impetigo

CARBUNCLE

Cellulitis
 LABORATORY DIAGNOSIS
Specimen:
 Pus, blood from septicaemic patient,

 respiratory secretions, urine,

 CSF, body fluids etc.,

Microscopy:
Gram stain- shows pus cells with GPC in groups

Culture :
 Blood agar, NA,
selective media:–
 mannitol salt agar

 Ludlam’s /salt milk agar

 incubated at 37°C for 24 hours.

 Pus may also be collected
Pus specimen may be collected
in a sterile container
on a swab
D test
 DRUG RESISTANCE
3 types

I) Production of beta lactamases

II) Alteration of penicillin binding protein (PBP),

Chromosomally mediated

III) Development of tolerance to the drug.

Penicillin resistance: Use of Methicillin, cloxacillin- against :

inhibitors of beta lactamase enzyme
Plasmid borne resistance : Erythromycin ,Tetracycline,Aminoglycosides

Methicillin Resistant Staph. Aureus (MRSA)

Vancomycin & teicoplanin- DOC
 Staphylococcus epidermidis

 Coagulase negative Staphylococcus present on normal

human skin

 Mostly causes opportunistic infections

 Predilection for growth on implanted foreign devices

like heart valves, catheters etc.
Staphylococcus epidermidis

 Produces ‘slime- bio flims’

 around the colonies
 prevents penetration of antibiotics & antibodies

 Causes stitch abscess, endocarditis & septicemia

 Usually resistant to multiple drugs.

 Staphylococcus saprophyticus

 Skin/urethral commensal
 Important cause of UTI in sexually active young women
 Usually sensitive to wide range of antibiotics

 Resistant to Novobiocin
 Coagulase -negative
 Mannitol - non fermented
 Phosphatase -negative
STREPTOCOCCUS
 Streptococcus

 Gram positive cocci in chains

 Part of normal human flora

 Important cause of pyogenic infections

Gram positive cocci in chains
 CLASSIFICATION OF STREPTOCOCCI
O2 requirement

Aerobic & facultative

anaerobes Obligate anaerobes

Hemolysis
Alpha hemolytic Non hemolytic
Viridans group Gamma hemolysis
Beta hemolytic
[Link] Enterococcus

20 Lancefield groups (A-V except I,J)

Eg:- [Link] ( group A )
[Link] ( group B )
[Link] ( group c )
etc Griffith’s types >80
 Classification (contd)

Lancefield classification is based on carbohydrate

(polysaccharide) antigen on the cell wall

 20 groups
 Group A Streptococcus pyogenes
 Serological typing based on M protein
 Griffith types(1-80)
Streptococcus pyogenes colonies showing beta hemolysis
Determinants of pathogenicity (Virulence factors)

 Capsular hyaluronic acid: antiphagocytic

 Cell wall polysaccharide:
 M protein: antiphagocytic, degrades C3b
 F protein: Mediates adherence to epithelial cells
 Pyrogenic exotoxins
 Streptolysin S: Lyses blood cells
 Streptolysin O: Immunogenic, Lyses blood cells
 Streptokinase: Facilitates spread of infection, lyses blood clot
 DNA’se: Depolymerises free DNA in purulent material
 Hyaluronidase: Helps spread of infection
 Infections caused by [Link]

I ) Suppurative infections:

a. Respiratory infections:
 Sore throat, tonsillitis
 Scarlet fever

b. Skin & soft tissue infections:

 Impetigo, pyoderma, erysipelas infection of wounds,
burns, & chronic skin lesions
 Respiratory infection

 Primary site –throat

 Tonsils or pharynx

 All serotypes

 Adherence by lipoteichoic acid

 Can spread to surrounding areas from throat

 Ludwigs angina, suppurative adenitis ,meningitis

Acute tonsillitis
Pharyngitis
 Impetigo & erysipelas (skin infections)

 Pyoderma , wound or burn infection

 Impetigo & erysipelas

 Erysipelas– diffuse infection involving skin, lymphatics- red

swollen , indurated– usually older people

 Impetigo—Small well circumscribed boils

 Caused by limited number of Strep seroytypes

 erysipelas
Impetigo
 Impetigo & erysipelas
(complications)

 Necrotizing fasciitis & cellulites

 Necrotizing fasciitis – by M types 1 &3

 Also called flesh eating bacteria

 Extensive necrosis of subcutaneous tissue & muscle with
severe systemic illness.
Other infections caused by [Link]

c. Genital infections
 Puerperal sepsis

d. Deep infections
 Bone & joint infections, lymphadenitis,
septicemia, acute endocarditis
 Non-suppurative complications of
Streptococcal infections

Rheumatic fever
 Primary site of infection is throat
 Marked immune response

Glomerulonephritis
 Primary site of infection is throat or skin
 Complement level is lowered
Diagnosis of Rheumatic fever

 H/O of fever with sore throat

 Joint pains ( large joints , fleeting pain)
 Small joints not involved
 Changing murmurs of the heart
 Nodules (subcutaneous nodules)
 Erythema marginatum – rash on trunk & arms
 ASO titer of >200 ( antibody to Streptolysin )
 CRP is raised
 Diagnosis Acute glomerulonephritis

 H/o of impetigo
 Puffiness of face , ↓ urine , albuminuria,
Microhaematuria
 > 200 ASO
 >300 anti DNase B (antideoxyribonuclease B)
 antihyaluronidase

 Scarlet fever

Scarlet fever is highly contagious and is

spread by sneezing, coughing, or direct
contact.

 signs of scarlet fever are the characteristic

rash, and the presence of a strawberry
tongue in children.

 One benzathine penicillin injection is

required for treatment.

 alternative antibiotics such as erythromycin

or clindamycin may be used
 LABORATORY DIAGNOSIS

Specimen:- Throat swab ,Pus, blood

Direct gram staining:- Gram positive cocci chains in short chains

Culture :- Pike’s transport medium,

5-10 %sheep blood agar
Anaerobic or with 5 to 10% carbon dioxide

Specific methods to confirm group A streptococcus

Bacitracin sensitivity
Rapid kits (latex agglutination)
Fluorescent antibody technique
Streptococcal colonies showing beta
hemolysis on Sheep Blood agar
Pus cells with Streptococci
Streptococcus pyogenes
showing Bacitracin sensitivity
Diagnosis of non- suppurative infections

 Serum sample
 Serological tests for detection of ASO
(anti-streptolysin O antibodies)
 Latex agglutination test – ASO titre 200 IU/ml
 Rise occurs after 2-3 weeks of initial infection
 Prognostic value
 Treatment

 All beta hemolytic Strep are Sensitive to Penicillin

G & Ampicillin & most are also Sensitive to
Erythromycin
 In case of Penicillin allergy , use Erythromycin
& cephalexin
 Tetracycline & Sulphonamides are not recommended
 Prophylaxis for Rheumatic fever
 2.4 mega units of Bezathine Penicillin X every 3
weeks
OTHER HEMOLYTIC STREPTOCOCCI
Human infections :

• Streptococci Group B, C, D, F, G
• Rarely H, K, O, R
 STREPTOCOCCUS AGALACTIAE
(GROUP B STREPTOCOCCI)

• Only species in Group B

• Gram positive cocci in pairs and short chains

• Morphologically similar to [Link]

• Commensal in genitourinary tract and lower gastrointestinal tract

• Vaginal /Rectal carriers – 30% seen in women

• Neonates- early onset/ late onset infections

 HUMAN INFECTIONS

• Causes septic abortion and puerperal sepsis

• Common infections -cellulitis, soft tissue infections, UTI,

pneumonia, endocarditis.

• Neonatal septicaemia and meningitis

 Infections in Adults
• Found with predisposing factors- DM, liver disease,
malignancy, renal failure
• Pregnant women – UTI after delivery.

• Puerperal infection of mother

– Upper genital tract; amniotic fluid, bacteremia,

• Primary bacteremia
•Pneumonia

Other infections:
• Arthritis Osteomyelitis
• Peritonitis SSTI
• Conjunctivitis
 LAB DIAGNOSIS
Specimen:- C.S.F, Ear swab, blood, High vaginal swab
Gram staining:- Gram positive cocci in short chains

Culture :-
Blood agar – Beta haemolysis
Catalase negative
CAMP TEST
Hippurate hydrolysis
Bacitracin –resistant
PYR negative
Orange pigment production – Islam’s medium
GRAM STAIN-
Gram positive cocci in chains
BLOOD AGAR- beta hemolysis
HIPPURATE HYDROLYSIS-
Positive
 CAMP TEST- POSITIVE
(Christie, Atkins, Munch, Peterson)
Enhanced hemolysis
near Staphylococcus

Streptococcus
agalactiae

Staphylococcus
aureus

Enterococcus
TREATMENT:- Penicillin G, Ampicillin. Alternatively
vancomycin, macrolides and cephalosporins.

25% -50% of neonates survive with permanent defects as

blindness, mental retardation.
 Group D

Enterococcal Non Enterococcal

[Link] & [Link]

 Possess the common group D lipoteichoic antigen

 Normal commensal of gastrointestinal tract, oral cavity,

gall bladder, urethra and vagina.

 Bile esculin hydrolysis test positive

 ENTEROCOCCUS
Classified earlier as group D
Can grow in
• 6.5% NaCl , 10° & 45°C
• hydrolyze bile esculin
• survive heating at 60 C for 30 min
• Survives pH 9.6

• PYR test - positive (Pyrrolidonyl- beta- naphthylamide)

• Based on sugar fermentation – 5 groups and has 16 species

• Most human infections from E. faecalis or E. faecium;

• Gram positive cocci in pairs and short chains

 MODE OF INFECTION

 Spread by fomites

 Nosocomial infections

 catheterization

 Major surgeries

 Wounds

 Cystoscopy
 CLINICAL MANIFESTATIONS
• UTI

• Bacteremia and mitral valve endocarditis (iv drug abusers)

• Intra abdominal , soft tissue and pelvic infections

• Late onset neonatal sepsis and meningitis

• Wound and tissue infections (burn surface)

 LAB DIAGNOSIS

Gram stain :- Gram positive cocci in pairs

Culture :-
Blood Agar- non haemolytic colonies (rare α and β)

Biochemical test
• Bile esculin hydrolysis test positive

• PYR test- positive

• Growth at- 6.5% Nacl, 40% bile,

• pH 9.6, 45° C & 10° C

Intrinsically resistant to cephalosporins

ENTEROCOCCUS-GRAM STAIN
 Enterococcus - Blood agar

2/8/2020
Bile esculin test
2/8/2020
TREATMENT

 Enterococci are intrinsically resistant to cephalosporins ,

cotrimoxazole and some penicillins.

 Acquired resistance encoded on plasmids and transposons.

 Combination therapy

 Vancomycin resistance is also noted, in such cases quinopristin

and dalfopristin can be given

 Non enterococcal species are sensitive to penicillin.

 Alpha Hemolytic Streptococcus

Features [Link] Viridans

streptococci

Haemolysis Alpha haemolytic Alpha

haemolytic

optochin Sensitive Resistant

Bile solubility positive Negative

 Streptococcus pneumoniae

 Pneumonia

 Bronchitis

 ENT infections (otitis media, sinusitis)

 Bacteremia

 Meningitis
 Pneumococci are the leading cause of pneumonia –
Lobar & Bronchopneumonia.

 Pneumococcal pneumonia has a high rate of

hospital transmission

 Route of entry – respiratory tract

 Source of infection: respiratory tract of carriers

 Mode of Transmission: inhalation

droplets/droplet nuclei/fingers
Carriers
II) Bronchopneumonia
III) Meningitis

 Most serious
 Secondary to pneumonia, otitis, sinusitis
 All age groups

IV) Other suppurative lesions:

 Empyema
 Pericarditis
 Otitis media
 Sinusitis
 Conjunctivitis
 Suppurative arthritis
 Peritonitis
Lab Diagnosis
Demonstration of Pneumococci by:

 Gram staining

 Culture

 Antigen detection

 Antibody demonstration
Specimens
 Sputum (acute lobar pneumonia)

 Pleural exudate (empyema)

Blood (acute lobar pneumonia /isolation from blood

implies bad prognosis)

 CSF (meningitis)

 Ear discharge etc

 Direct smear –
Microscopy  Gram positive lanceolate

 diplococci , plenty of
polymorphs

 Capsulated (clinical
samples), Non motile

 Capsule demonstrated by
India ink
 Negative
staining
 Blood Agar: Colonies - small
dome shaped alpha
hemolysis around them.

Further incubation – flat with

raised edges, circular with

central depression.

- typical DRAUGHTSMAN

or CARROM COIN appearance.

 Biochemical reactions

 Ferment variety of serum sugars

particularly INULIN with acid production

Bile solubility test is positive with 10%

sodium deoxycholate

 Sensitive to Optochin (ethyl hydro cuprein)

 Catalase & Oxidase negative

 Antigenic Properties
[Link] polysaccharide or Specific soluble substance (SSS)

90 serotypes based on antigenic nature

Typing methods

1. Agglutination test

2. Precipitation of SSS with antiserum

3. Quellung reaction
 Treatment and Prophylaxis

 Penicillin (parenteral route) or Amoxycillin

Penicillin resistant cases (resistant to erythromycin and

tetracycline) can be treated with third generation

Cephalosporins

Vaccine – polyvalent highly purified capsular

polysaccharide extract from 14 prevalent serotypes.

 Strep viridans

Dental carries – [Link] ( sucrose- acid & dextran)

Thank you