Gram Positive Cocci
Gram positive Staphylococci
Objective
• To know the general characters of the genus Staphylococcus
• To know how to differentiate between staphylococci and other gram-positive cocci
• To know the virulence factors associated with staphylococci
• To know the clinical infections associated with staphylococci
• To know the differential tests that used to identify the clinically relevant staphylococcus species
• To know the methicillin resistance in a serious of clinical problem
STAPHYLOCOCCI
Staphyle (Greek) Bunch of grapes or BerriesCocci (Rounded)
General Characteristics•Gram positive cocci Arranged in grape like clusters •Facultative anaerobic •Catalase-positive•Grow in ordinary culture media (Nutrient agar and broth)
Species of Staphylococci
33 species known
Three are medically important:
•S. aureus
•S. epidermidis
•S. saprophyticus
STAPHYLOCOCCUS AUREUS
Morphology & Cultural Characters
• Staphyle - bunch of grapes
• Aureus - golden color colonies
• On blood agar - beta-hemolytic colonies
In contrast to other Staphylococci Can grow in 7.5% NaCl & basis of mannitol
ferment mannitol salt agar as selective medium for S.aureus
Staphylococcal cell wall structure
Capsule orpolysaccharideslime layer
Phospholipid
PBP PBP
Transport protein
Cytoplasmic membrane
Protein A
Peptidoglycanlayer
Techoic acid
Capsule Antigenic & Antiphagocytic
Teichoic acid •Binds to fibronectins on surface of host cells
Protein AHas great affinity to Fc portion of IgG and makes it unavailable to its receptors on phagocyte
Coagulase
Fibrin ClotFibrinogen
EXTRACELLULAR ENZYMES
The clot coats the organisms and inhibit their phagocytosis
1. Coagulase
2. Catalase
May prevent killing of S. aureus by PMNLs cells
EXTRACELLULAR ENZYMES
Catalase
H2O2 Water O2+
EXTRACELLULAR ENZYMES
3. Lipase
4. Hyaluronidase
5. Protease
6. DNAase
7. Penicillinase
TOXINS OF S. AUREUS
1. Cytolytic toxins (Haemolysins & Leukocidins)
A- Haemolysins (hemolyzing RBCs)
, hemolysin: hemolyzing RBCs and destroying platelets
, hemolysin: als known as hot-cold lysin, enhances haemolytic activity when incubated at 370 C and 40C
δ hemolysin: less lethal than and tHaemolysins.
B-Leukocidins
Is an exotoxin lethal to PMNs also it has been implicated to as contributing to invasiveness of the organisms by suppressing phagocytosis
2. Exfoliatins (epidermolytic)
Cause nerosis of epidermis (locally or at other sites)
TOXINS BY S. AUREUS
3. Toxic shock syndrome toxin (TSST-1)
•Stimulates macrophages to produce IL-1 & TNF-alpha
•Multiple effects toxic shock
4. 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 by S. aureus
PyogenicDue to toxins
Superficial Deep
S. AUREUS DISEASESI: PYOGENIC INFECTIONS
A. Superficial
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 bacteremia
Furuncle Folliculitis
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
S. AUREUS DISEASES
I: PYOGENIC INFECTIONS
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
S. AUREUS DISEASES
S. AUREUS DISEASES II: DISEASES DUE TO TOXINS
Exfoliatin inStaph lesion
Toxemia
Necrosis of epidermis
Loose skin(vesicles)
Vesicles breakup
Staph not isolated from lesions
1. Scalded Skin Syndrome (SSS)
Staphylococcus Scalded Skin Syndrome (SSSS)
2. Impetigo •Localized SSS •Produce blisters that contain pus and Staph
3. Scarlet Fever•Mild form of SSS•Characterized by erythema without vesicles
S. AUREUS DISEASES II: DISEASES DUE TO TOXINS
4. Toxic Shock Syndrome (TSS)
• Occur in young women during and immediately after menstruation
• Due to intravaginal use of tampons infected with S. aureus produce TSS
• Fever, vomiting, diarrhea, body pain within 48 hours severe shock
S. AUREUS DISEASES II: DISEASES DUE TO TOXINS
Staph multiplication
Enterotoxinin food
Resists reheating
Acute vomiting &diarrhoea (1-5 hrs)
Food ingested
5. Staph Food Intoxication
Poorreferigeration
Staph Carrier
Salads, potatocreamy dishes
Contaminates
S. AUREUS DISEASES II: DISEASES DUE TO TOXINS
TREATMENT OF STAPH INFECTIONS
Drainage of pus in superficial and chronic lesions
C/S to select proper antibiotics
Severe infections
Amoxycillin-clavulinic acid (Augumentin)
Chronic infections
MRSA (methicillin-resistant S. aureus)
Vancomycin
VRSA (Vancomycin Resistant S.aureus) &
VISA (Vancomycin Intermediate S.aureus)
Linezolid & Quinupristin-Dalfopristin
•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) was introduced•In 1961 methicillin-resistant S. aureus (MRSA) was discovered (presently a major problem)•In 1996 Vancomycin Intermediate S. aureus (VISA)•In 1997 Vancomycin Resistant S. aureus (VRSA)
ANTIBIOTICS RESISTANCE Historical aspect
STAPHYLOCOCCAL NASAL CARRIER
Anterior nose of 20-40% of adults are carriers•Physicians & nurses = 50-70% •Also skin of axillae & perineum•In hospital - high carrier rate 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
Control of Carrier and Re-infection
•Wash clothes in hot water (>70oC)
•Hand washing with 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. epidermidis2. S. saprophyticus
STAPHYLOCOCCUS EPIDERMIDIS
•Normal flora in °Skin
°Anterior nose &
°External ear canal
•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
LAB IDENTIFICATION OF S. AUREUS
Specimens
• Pus, sputum
• Blood, CSF
• Feces, vomit and left over food – in food poisoning
• Anterior nasal swab for carriers
LAB IDENTIFICATION OF S. AUREUS
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)
Sputum smear Staphylococcus aureus
Staphylococcus & Streptococcus pneumoniae
LAB IDENTIFICATION OF S. AUREUS
Biochemical Tests
Catalase TestDifferentiates between Staphylococci (positive) & Streptococci (negative)
H2O2 Water + OxygenCatalase
•Pour 2-3 ml of H2O2 in a test tube
•With a sterile wooden stick pick a good growth of the
organism (from blood free medium) and immerse in H2O2
•Immediate active bubbles – positive test
LAB IDENTIFICATION OF S. AUREUS
Biochemical Tests
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
Coagulase
Activates
CRF* (Prothrombin) in plasma
*CRF : Coagulase Reacting Factor
Clot FibrinogenFibrin
Activated CRF
1. Coagulasea) Free coagulase (soluble):
•Converts fibrinogen to fibrin -clot formation•The clot coats the organisms and inhibit their phagocytosis•Detected by tube coagulase test (positive in 3-4 hours)
Coagulase
Fibrin ClotFibrinogen
EXTRACELLULAR ENZYMES
•Converts fibrinogen to fibrin -clot formation•The clot coats the organisms and inhibit their phagocytosis•Detected by slide coagulase test (positive in few min)
2. Bound coagulase
LAB IDENTIFICATION OF S. AUREUS
Biochemical TestsTube 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)
LAB IDENTIFICATION OF S. AUREUS
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.
LAB IDENTIFICATION OF S. AUREUS
DNASE TEST•Differentiates 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
Case study #1
An elderly male was admitted to CCU with myocardial infarction. He was helped with IV lines and urinary catheter. On 5th day of his stay, he was found to have :
1. High grade temperature. 2. The physician noticed redness around IV
line.3. Chest X-ray showed normal lung fields. 4. Blood culture showed gram-positive cocci
in clusters5. Growth on blood agar had coagulase-
negative 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 will you do if the organism is
resistant to vancomycin ?
Case study #2
An 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 ?
Case study #3
A19-year-old women complained of fever and flank pain, dysuria, urgency to urinate, and blood-tinged urine were also noted. A urine analysis revealed many WBCs and WBCs cast. A urine culture grew white nonhemolytic colonies on blood agar. The colony count was 45,000 CFU/ml. No growth appeared on MacConkey’s agar. The organism was catalase positive and slide-and tube coagulase negative and produced a 21-mm zone of inhibition in the presence of novnbiocin disc
Questions
•What is the identity of the isolate?
In what patient population does this organism normally cause infection?
Top Related