Diagnostic microbiology lecture 8 THE GRAM POSITIVE COCCI
Diagnostic microbiology lecture: 8 THE GRAM POSITIVE COCCI Abed El. Kader Elottol MSc. Microbiology 2010 1
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STAPHYLOCOCCI 3
Species 33 species are known. Three are medically important: 1. Staphylococcus aureus 2. Staphylococcus epidermidis 3. Staphylococcus saprophyticus 4 Most important pathogen May cause endocarditis May cause cystitis.
General Characteristics 1. Cocci arranged in grape-like clusters 2. Strongly gram-positive 3. Ferments many carbohydrates with the production of lactic acid but no gas 4. Non-motile 5. Non-spore forming 5
Staphylococcus aureus • Aureus: golden color (golden color colonies on blood agar) • Diseases caused by the organism: 1. Toxic shock syndrome 2. Furuncles (abscess) 2. Septicemia 4. Impetigo 5. Meningitis 6. Pneumonia 7. Food poisoning 8. Pyoderma 6
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Cultural and Morphological Characteristics 1. Media for Primary Isolation: S. aureus grow well in most routine media e. g, Blood Agar, Nutrient Agar. 2. Media for Selective Isolation: Mannitol Salt Agar (MSA), is an excellent medium which contains 7. 5% sodium chloride which is considered as a high percentage and inhibitory to most medically important bacteria. In addition it contains mannitol as the only carbon source and a p. H indicator to detect mannitol fermentation by S. aureus. 3. Incubation: After streaking the specimen on one of the common media, incubate the plates at 35 -37 o. C for 24 hours. 8
Colony Morphology • On blood agar plates: colonies are 2 -4 mm in diameter, rounded and slightly elevated. Most pathogenic strains produces a zone of β-hemolysis. Another distinguishing character is the production of a golden yellow pigment. • On Mannitol Salt Agar: The colonies are surrounded by a yellow zone indicative of acid production resulting from the fermentation of Mannitol. 9
Gram Stained Smears: • Gram-positive cocci arranged in clusters. • Single cells, diplococci, and short chains may also appear. • It is usually simple to identify the morphology in stained film from sputum or pus but one can be certain by performing simple biochemical tests for the isolate e. g. , • catalase test to differentiate it from Streptococci and Coagulase or DNase to differentiate it from non-pathogenic staphylococci. . 10
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Biochemical Characteristics 1. Catalase positive 2. Mannitol fermenter 3. Grow well in 7. 5% Na. Cl 4. Coagulase positive 5. DNase positive 6. Glucose fermenter. 12
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Identification: 1. Based on gram-staining 2. To differentiate it from other gram-positive cocci (Streptococci) perform catalase test 3. To differentiate it from other non-pathogenic staphylococci a group of biochemical tests are performed. a. Coagulase test b. Mannitol fermentation c. Growth in 7. 5% Na. Cl d. Glucose OF e. DNase 14
PHAGE TYPING • A Staphylococcus aureus-specific phage is added to a plate that is inoculated with S. aureus. • The plates are incubated at 37 o. C for 24 hours. • Positive identification: Formation of plaques • Negative results: S. aureus grow over the whole area. 15
Sensitivity testing and treatment • S. aureus is a frequent hospital pathogen and it has the ability to develop resistance to the commonly known antibiotics. • For this reason sensitivity testing must be performed on all isolates. • Penicillin G and its derivatives (ampicillin, amoxycillin, cloxacillin, methicillin), ofloxacillin and cephalosporins are usually effective against S. aureus. 16
ANTIBIOTICS RESISTANCE Historical aspect • 1940 s : all S. aureus were sensitive to penicillin • Shortly after use : penicillin resistant strains appeared which produced betalactamase rapidly spread • In late 1950 s : beta-lactamase - resistant penicillin (methicillin) (not degraded by) • In 1961 methicillin-resistant S. aureus (MRSA) was discovered (presently a major problem) 17
MRSA • Low carriage rate in community • High in tertiary care hospitals Mode of Transmission • Fomites • Direct from hospital staff or attendants : contaminated hands 18
• MRSA causes a variety of disseminated, lethal infections in humans. • Has the ability to easily transfer resistant genes to other species directly and indirectly. • Overuse of antibiotics imposes selective pressures which mediates the acquisition of resistance. • Most major organs fail with disseminated MRSA. 19
PREVENTION OF STAPH INFECTIONS Control of Carrier and reinfection Wash clothes in hot water (>70 o. C) 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 • • 20
Negative Oxidase test
COAGULASE-NEGATIVE STAPHYLOCOCCI (CNS) 22
• 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) 23
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) 24
Staphylococus saprophyticus 25
• Saprophytic in 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 26
The End 27
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