staphylococci - prac. microbiology

Staphylococci

Morphology

Gram positive cocci

in clusters

(streptococci catalase –ve)

Catalase: +ve Coagulase:

+ve: S. aureus-ve: CoNS

Coagulase Test

Principle:

FibrinogenPlasma

Coagulase enzyme FibrinVisible Clot

Procedure:2

3 Incubate at 37oC, observe for formation of visible clot.

1 ml dil. citrated plasma1

Broth culture

(organism)

S.aureus

(coagulase +ve staphylococci)

Morphology

Gram positive cocci

in clusters

CultureNutrient agar (golden yellow endopigment)

Blood agar (ß-haemolysis)

Mannitol-Salt agar (MSA)

• Selective, differential medium.

• 7.5% sodium chloride + mannitol sugar.

S. aureus (ferment)

• Mannitol Acid

(yellow)(yellow)

CoNS (No fermentation)

• Mannitol No acid

(Red)(Red)

Virulence Factors for Pyogenic Diseases

A- Localized Skin infectionsPYOGENIC DISEASES

B- Staphylococcal Pneumonia

C- Invasive Infections

• Bacteremia.• Osteomyelitis.• Acute bacterial endocarditis.• Meningitis.• Septicemia.

Diagnosis of Pyogenic Diseases

1- Specimen:

Abscess ----- pus Endocarditis ----- blood

Meningitis --- CSF, blood Pneumonia ----- sputum

UTI ----- urine

2 -Direct detection

3 -Culture

Other than blood

(pus, CSF, urine)

Blood

subculture

4 -Identification

A- Colony Morphology

B- Microscopy

C- Biochemical Reactions

1-Coagulase 2-Clumping factor

3-Catalase

Toxin-Mediated Diseases

A- SSS Exfoliatin

B- TSS TSST-1

C- Food poisoning Enterotoxins

A- Staph. Scalded Skin Syndrome

B- Toxic Shock Syndrome Predisposing factors:- First observed in Menstruating females using tampons )tampon retained in vagina for 4-5 days(- Any staphylococcal lesion can predispose to TSS.

Virulence factor: TSST-1

Diagnosis: clinical picture.culture: for isolation & identification.

ELISA: for toxin detection in blood

C- Staph. aureus food poisoning

I.P: 1- 6 hrs.

Clinical picture:Colic, vomiting, mildDiarrhea, but no fever

Diagnosis:1. Specimen: food, vomitus, stools.

2. Culture: on MSA.

3. ELISA: for toxin detection in sample or in isolated organism.

4. Strain typing: to trace source of infection )e.g. phage typing(.

Strain Typing

• Phage typing.• Colony morphology.• Biotyping.• Plasmid profile.• Ribotyping.• Chromosomal

analysis.

Treatment1 -Penicillin-resistant S.aureus

2 -MRSA.

3 -VISA

VRSA

Coagulase negative Staphylococci (CoNS)

• General Characters:– Non-hemolytic on blood

agar– Coagulase negative– Mannitol non-fermenter

• Diseases: S. epidermidis:– Device-related infections

(prosthetic valve endocarditis, shunt infections, … etc…)

– Urinary tract infection– Wound infection

S. saprophyticus: UTI )honeymoon( cystitis

Case 1 A man underwent an emergency operation for

perforated appendix. He required admission to the Intensive Care Unit. On the 5th day of hospitalization he developed fever, and the wound was erythematous, swollen, with purulent discharge )Surgical site infection “SSI”(. The surgeon requested bacteriological examination )culture and sensitivity( of the wound pus

Surgical site infection

Specimen

• Wound swabs were obtained.

• Gram staining & Microscopy Gram-positive cocci arranged in clusters amongst pus cells.

On nutrient agar →

Golden yellow pigment

On Blood agar →Beta-hemolysis

Culture

On MSA →Yellow colonies

Identification:Film from colonies Gram-positive cocci in clusters

Biochemical reactions

Catalase +ve Coagulase +ve Clumping factor +ve

Which organism is causing this condition?

• Gram-positive cocci in clusters, catalase positive

staphylococci

• β-hemolysis , golden yellow pigment, yellow colonies on MSA and coagulase positivity

S. aureus

.

Antimicrobial susceptibility “Disc diffusion” the organism is resistant: to oxacillin, penicillin,erythromycin, & gentamicin but sensitive to: vancomycin.

How do you interpret the susceptibility data?

The organism is multi-resistant, including oxacillin-resistant

methicillin-resistant S. aureus (MRSA).

Name other diseases caused by this organism.

1. Pyogenic infections a. Localized skin infections, e.g. folliculitis, furuncles,

abscess. b. Staph pneumonia c. Invasive staph. infections, e.g. endocarditis, cellulitis,

osteomyelitis, and septic arthritis

2. Toxin-mediated diseases a. Toxic shock syndrome , b. Scalded skin syndrome,

c. Food poisoning.

A group of people shared a carbohydrate- rich meal. 1-6 hours later, they manifested vomiting, diarrhea but no fever. The condition was mild and self- limited.a( What could be the clinical diagnosis?b( What is the possible causative organism?c( What is the virulence factor responsible for this condition?d( What is the mechanism of action of such virulence factor?e( What is the laboratory test that is used to trace the source of this outbreak?

Case 2

MCQs

1. All the following are characteristic features of staphylococci except:

a) Gram positive cocci.b) Arranged in clusters.c) Catalase negative .d) Usually grow on simple media.e) Usually inhabit skin and mucosa.

2. The following medium is used to isolate S. aureus from specimens contaminated by other bacteria: a) Nutrient agar. b) MacConkey's agar. c) Blood agar . d) Mannitol salt agar. e) Chocolate agar

3. The localized nature of S. aureus lesions is due to:a) Adhesins. b) Protein A.c) Staphylocoagulase d) Staphylokinasee) Exfoliatin

4. S. aureus growth can be identified by the following characters except:a) Colony on blood agar is golden yellow.b) Colony on blood agar is surrounded with a clear zone. c) Colony on Mannitol salt agar is rose pink.d) Protein A positive. e) Coagulase test positive.

5. One of the following is a S. aureus toxin-mediated disease: a) Folliculitis b) Staphylococcal pneumonia. c) Toxic shock syndrome. d) Osteomyelitis e) Carbuncles

6. Staphylococcal strain typing is used :

a) To diagnose staphylococcal pyogenic diseases.

b) To differentiate between coagulase -ve and coagulase +ve staphylococci.

c) To differentiate between staphylococci & streptococci.

d) To trace the source of outbreaks of S. aureus in the epidemiological studies.

e) To identify enterotoxin-producing strains of S. aureus.