Staphyle (Greek) Bunch of grapesCocci Berries

•Gram positive cocci arranged in grape like clusters•Aerobic and facultative anaerobic
•Catalase-positive
•Grow in ordinary culture media nutrient agar and broth)

species known three are medically important:
• S. aureus
• S. epidermidis
• S. saprophyticus


STAPHYLOCOCCUS AUREUS Morphology
• Staphyle - bunch of grapes
• Aureus - golden colour (golden colour colonies on blood agar)
• Gram +ve cocci in clusters
• Some strains capsulated

CULTURAL CHARACTERISTICS
• Aerobic and facultative anaerobic
• Can grow on ordinary media like nutrient agar
• On blood agar – golden yellow colonies with beta-haemolysis
• In contrast to other Staphylococci ;

Can grow in 7.5% NaCl & basis of mannitol as
salt ferment mannitol selective medium for S. aureus


ANTIGENIC STRUCTURE
Teichoic acid (ribitol type)
• group specific antigen
• binds to fibronectin on surface of host cells
Protein A • group specific antigen
• has great affinity to Fc portion of IgG and makes it unavailable to its receptors on phagocyte.
Capsule
• antigenic & antiphagocytic




EXTRACELLULAR ENZYMES


coagulasefibrinogin_fibrin_clot


catalase

H2O2

























3. Lipas
• Hydrolyses lipids in cutaneous and subcutaneous tissues  invasiveness
4. Hyaluronidase (spreading factor)
• Breakdown hyaluronic acid in CT  invasiveness
5. Protease
6. DNAase
7. Penicillinase

TOXINS S. AUREUS

1. Haemolysins: (alpha, beta, gamma, delta)
Alpha-haemolysin
• Exotoxin
• Lethal to RBCs, WBCs and platelets
2. Leukocidins
• Exotoxin distinct from haemolysins
• Attack WBCs
3. Exfoliatins (epidermolytic) :
• Cause nerosis of epidermis (locally or remotely)
4. Toxic shock syndrome toxin (TSST-1)
• Stimulates macrophages to produce IL-1 & TNF
• Multiple effects  toxic shock
5. Enterotoxins
• Resist boiling for 30 min.
• Resist gastrointestinal enzymes.
• Act on neuronal receptors in upper GIT
• Cause gastroenteritis in 1-5 hours after ingestion



















DISEASES CAUSED BY S. AUREUs
I: SUPPURATIVE (pyogenic) INFECTIONSA. Superficial Infections
1. Furuncle (boil)
• Infection of hair follicle, sweat gland, sebaceous glands
• Blockage of ducts predispose to infections like acne vulgaris & stye
2. Carbuncle• (Furuncle + Inflammation of subcutaneous tissue)
• Can lead to abscess formation and bacteraemia
3. Paroncyhia
• Infection of nail bed
• Can be autoinfection OR from an external source
4. Postoperative wound infections• Autoinfection or from carriers like doctors and nurses
5. Nosocomial (hospital acquired) infections : A common cause




B. Deep Infections Usually caused by bacteremic spread
1. Osteomyelitis and arthritis
3. Bronchopneumonia
4. Empyema thoracis
5. Meningitis
6. UTI
7. Endocarditis: in drug abusers using injections

II: DISEASES DUE TO TOXINS
1. Scalded Skin Syndrome (SSS)













2. Bullous Impetigo • Superficial skin infection due to exfoliatin
• Produce blisters that contain pus and Staph
• Direct spread can occur (localized SSS)
• Infants and children usually affected
3. Scarlet Fever• Mild form of SSS
• Characterized by erythema without vesicles


4. Toxic Shock Syndrome• Occur in young women during and immediately after menstruation
• Due to intravaginal use of tampons infected with S. aureus  produce TSST–1
• Fever, vomiting, diarrhoea, body pain within 48 hours  severe shock
• Skin rashes deeper than SSS may appear
5. Staph Food Intoxication















TREATMENT OF STAPH INFECTIONS• Drainage of pus in superficial and chronic lesions
• C/S to select proper antibiotics
Infections Treatment
a) Severe infections PRP* (cloxacillin,methicillin)
b) Due to MRSA Vancomycin (Final choice)
(methicillin-resistant S. aureus)
c) Chronic infections PRP (given for months)
*PRP : Penicillinase resistant penicillins


ANTIBIOTICS RESISTANCE Historical aspect
• 1940s : all S.aureus were sensitive to penicillin
• Shortly after use : penicillin resistant strains appeared
which produced beta-lactamase - rapidly spread
• In late 1950s : beta-lactamase - resistant penicillin
(methicillin) (not degraded by)
• In 1961 methicillin-resistant S. aureus (MRSA)
was discovered (presently a major problem)

Antibiotic resistance of S.aureus













EPIDEMIOLOGY OF STAPH INFECTIONSSource of Infection (Carrier)
• Anterior nose of 20-40% of adults
• Physicians & nurses = 50-70%
• Skin of axillae & perineum
• In hospital - high due to environmental load
MRSA
• Low carriage rate in community
• High in tertiary care hospitals
Mode of Transmission• Fomites
• Direct from hospital staff or attendants : contaminated hands



PREVENTION OF STAPH INFECTIONSControl of Carrier and reinfection
• Wash clothes in hot water (>70oC)
• Use antiseptic soap (Dettol soap)
• Antimicrobial nasal cream (Gentamicin,Mupirocin)
• Oral antibiotics that are concentrated in nasal secretions (ciprofloxacin and rifampicin)
Chemoprophylaxis
• Antibiotics before and at time of surgical operation

COAGULASE-NEGATIVE STAPHYLOCOCCI• Medically important species:
• 1. S. epidermidis
• 2. S. saprophyticus

STAPHYLOCOCCUS EPIDERMIDIS• Normal flora in
° Skin,
° Anterior nose &
° External ear canal
• Cell wall contains teichoic acid (glycerol type)
• White, non-haemolytic colonies on blood agar
• Sensitive to novobiocin; (S. saprophyticus is resistant)

DISEASES BY S. EPIDERMIDIS• Most infections are hospital acquired
• Opportunistic pathogen in immuno-suppressed
• Strongly associated with presence of foreign bodies
° Prosthetic heart valves (endocarditis)
° IV catheters (bacteremia)
° Urinary catheter (UTI in elderly)
° CSF shunts (meningitis)
° Peritoneal dialysis catheter (peritonitis)


STAPHYLOCOCUS SAPROPHYTICUS• Saprophytic life
• Resistant to novobiocin
• Most infections are community-acquired
° Primary UTI in 10-20% of young adult women – hormonal factors may be involved.
• Resistant to antibiotics – penicillins & cephalosporins

A PROBLEMAn elderly male was admitted to CCU with myocardial
infarction. On 5th day of his stay, he was found to have :
1. High temperature with cough and purulent sputum
2. Chest X-ray showed multiple abscesses
3. Sputum Gram-smear showed Gram-positive cocci in clusters
4. Growth on blood agar had coagulase-positive bacteria
Questions
1. What is the identity of the organism ?
2. What type of infection it is ?
3. What is the source of organism ?
4. What is antibiotic treatment of the patient ?
5. What you will do if the organism is MRSA ?

LAB IDENTIFICATION OF S. AUREUS Specimens
• Pus, sputum
• Blood, CSF
• Faeces, Vomit and left over food – in food poisoning
• Anterior nasal swab for carriers
Microscopy• G+ve cocci in clusters
Culture
• Blood agar - golden yellow colonies with
beta-haemolysis
• Mannitol salt agar - yellow colonies (selective and differential for isolation from faeces)


Biochemical TestsCatalase Test
Differentiates between Staphylococci (positive) & Streptococci (negative)

H2O2 Water + Oxygen









Coagulase Test (positive)
• Differentiates between S. aureus (positive) and other staphylococci (negative)
Slide Method (for bound coagulase)
• Place a drop of saline on two separate slides.
• Emulsify a colony of test organism in each drop to make thick suspension.
• Add a drop of plasma to one suspension and mix gently.
• Look for clumping within 10 sec for positive test.

EXTRACELLULAR ENZYMES



















Tube Method (for free coagulase)1. Mix 0.2 ml of plasma with 1.8 ml of saline (1:10 dil)
2. Take three small test tubes and label
• T (Test organism) = (18-24 hr broth culture)
• Pos (Positive control) = (18-24 hr broth culture)
• Neg (Negative control) = (Sterile broth)
3. Pipette 0.5 ml of diluted plasma into each tube
4. Add 5 drops of test organism in tube “T”
5. Add 5 drops of S. aureus culture to tube “Pos”.
6. Add 5 drops of sterile broth to tube ‘Neg’
7.Mix gently and incubate at 37oC for 1 hr.
8. Examine for clotting each hr : upto 6 hours.

DNASE TESTDifferentiates S. aureus (positive)) from other Stpahylococci
(negative)
• Culture test organism DNA agar (S. aureus will hydrolyse DNA around the colonies)
• After 24 hrs incubation pour weak HCl on surface of plate
• The acid will ppt. unhydrolyzed DNA and DNAse producing colonies are surrounded by clear areas within 5 min.
• Clear zones: S. aureus – DNAse +ve
• No clearing around colonies – DNAse -ve



Phage Typing• Depends on lysis of staph by specific bacteriophages
• Typed into four phage groups (I, II, III, IV)
• The test is of epidemiological vlaue
• No diagnostic or therapeutic significance
1. Culture the organism on a marked plate (squares)
2. Place a drop of each phage in a specific square
3. Incubate for 24 hours
4. Clear zones of lysis around the specific bacteriophage

Lytic groups & subtypes of staphylophages
Groups Subtypes
I 29 52 52A 79 80
II 3A 3B 3C 55 71
III 6 7 42E 47 53
IV 54 75 77 83A 42D
Not 81 187