Diagnostic microbiologylecture: 8

THE GRAM POSITIVE COCCI

Abed ElKader Elottol MSc. Microbiology

2010

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Species

33 species are known. Three are medically important:1. Staphylococcus aureus Most important pathogen2. Staphylococcus epidermidis May cause endocarditis3. Staphylococcus saprophyticus May cause cystitis.

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General Characteristics

1. Cocci arranged in grape-like clusters2. Strongly gram-positive3. Ferments many carbohydrates with the production of lactic

acid but no gas4. Non-motile5. Non-spore forming

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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. Impetigo5. Meningitis 6. Pneumonia7. Food poisoning 8. Pyoderma

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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 pH 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 oC 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.

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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..

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Biochemical Characteristics

1. Catalase positive2. Mannitol fermenter3. Grow well in 7.5% NaCl4. Coagulase positive5. DNase positive6. Glucose fermenter.

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Identification:

1. Based on gram-staining2. To differentiate it from other gram-positive cocci

(Streptococci) perform catalase test3. To differentiate it from other non-pathogenic

staphylococci a group of biochemical tests are performed.

a. Coagulase testb. Mannitol fermentationc. Growth in 7.5% NaCld. Glucose OFe. DNase

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PHAGE TYPING

• A Staphylococcus aureus-specific phage is added to a plate that is inoculated with S.aureus.

• The plates are incubated at 37 oC for 24 hours.

• Positive identification: Formation of plaques

• Negative results: S. aureus grow over the whole

area.

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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.

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ANTIBIOTICS RESISTANCE

Historical aspect

• 1940s : all S. aureus were sensitive to penicillin• Shortly after use : penicillin resistant strains appeared

which produced betalactamase 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)

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MRSA• Low carriage rate in community• High in tertiary care hospitalsMode of Transmission• Fomites• Direct from hospital staff or attendants : contaminated hands

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• 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 .

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PREVENTION OF STAPH INFECTIONS

• Control 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

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Negative

Oxidase test

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COAGULASE-NEGATIVE STAPHYLOCOCCI (CNS)

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• 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)

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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)

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Staphylococus saprophyticus

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• 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

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The End