Staphylococci 09 10 Med

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?