Post on 02-Jul-2015
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Gram positive and Gram negative cocci
- brief overview of laboratory diagnosis -
Gram positive cocci
• Family: Micrococaceae• Genera:
– Staphylococcus– Micrococcus– Stomatococcus– Planococcus
• Family: Streptococacceae• Genera:
– Streptococcus– Enterococcus– Aerococcus– Gemella– Leuconostoc– Pediococcus– Lactococcus
Genus Staphylococcus
• Cocci: – Round shape; cluster arrangement (”grape-shaped”)– Gram positive – Aerobic growth (+anaerobic)– Nonsporulated
• Clinically significant microbial species:– S.aureus – pathogenic – S.epidermidis – accidentally pathogenic– S.schleiferi, S.lugdunensis, S.haemolyticus, S.saprophyticus –
low pathogenic potential
Staphylococcus aureus
• Community & Hospital acquired infections:
– Skin & subcutaneous tissues: foliculitis, abscesses, furuncles, carbuncles
– otitis, synusitis, pneumonia– Osteomyelitis, septic arthritis– Endocarditis, phlebitis, sepsis– Food poisoning– Meningitis, encephalitis
Laboratory diagnosis of Staphylococcal Infections: Collection of specimens
Pus:
Closed lesions (abscesses): • surgical collection:
– rigurous cleaning and disinfection of skin (iodine)
– Incision and aspiration of pus
Open lesions:• Cleaning and disinfection of skin around lesion (iodine)• Collection of pus with sterile swab / loop
Staphylococcus aureus: creamy, yellow pus
Celulitis with Staphylococcus aureus
Laboratory diagnosis of Staphylococcal Infections: Collection of specimens
Fluid from cavities
e.g. spinal (CSF)/ pleural / pericardic / articular• Sample collected by punctioning the cavity
• E.g. Lumbar punction (spinal tap) – patient lies on the side, knees pulled up toward chest,
chin tucked downward
– back cleaned and disinfected + local anesthetic – spinal needle inserted into lower back area– needle properly positioned, CSF pressure measured
and sample collected in sterile tube
Laboratory diagnosis of Staphylococcal Infections: Collection of specimens
Pharyngeal, naso-pharingeal exudate
Patient: – in the morning, before feeding, before brushing teeth;
alternatively: at least 4 hours since last meal & teeth brushing
– No mouth rinse, no chewing gum!
– No antibiotics during the last 7-10 days
Medical staff:– Wear gloves, face protection (mask, eye
protection/face shield), protective lab coat
Collection of pharyngeal exudate
• Dacron or Rayon swab• Tongue blade & good light• Insert swab behind uvula
without touching it
• Swab tonsils, posterior
pharynx + lesions (if any)• Avoid touching tongue,
cheeks, teeth• Place swab in sterile tube
• Transport to lab (RT/2-8°C)
Collection of pharyngeal exudate
Laboratory diagnosis of Staphylococcal Infections: Gram stained smear
• Gram positive cocci• Shape: spherical• aglomerated in clusters / pairs / isolated• Location: both intra- and extracellular
• Size: 0.5-1 µM
• + WBC (entire & destructed)
Staphylococcus: Gram staining biological product (sputum)
Staphylococcus: Gram staining
Laboratory diagnosis of Staphylococcal Infections: Innoculation of culture media
• closed collections / moderately contaminated collection sites (e.g. nasopharingeal swab)
↓blood agar
• S.aureus: round colonies, 1-3 mm diameter, smooth, hemolytic, pigmented (golden-yellow)
• S.epidermidis: white colonies
”Golden” colonies: Staphylococcus aureus
Laboratory diagnosis of Staphylococcal Infections: Innoculation of culture media
• closed collections / moderately contamnated collection sites (e.g. nasopharingeal swab) → blood agar
• Highly contaminated biological products (e.g. stool)
↓
Chapman agar - selective medium
(high salt content + mannitol + pH indicator)
WHY?:– A. Inhibit other germs, favour growth of Staphylococcus– B. Staphylococcal growth →Fermentation of mannitol →colour of
medium changes from pink to yellow (further identification step)
Mannitol Salt Agar (Chapman)
- high salt concentration supports growth of Staphylococcus / inhibits Streptococcus
- mannitol acidification - turn the medium colour to yellow
Chapman agar – mannitol acidification
Gram positive cocci
• Family: Micrococaceae• Genera:
– Staphylococcus– Micrococcus– Stomatococcus– Planococcus
• Family: Streptococacceae
• Genera:– Streptococcus
– Enterococcus
– Aerococcus– Gemella– Leuconostoc
– Pediococcus
– Lactococcus
Genus Streptococcus
• Clinically significant microbial species:– Streptococcus pyogenes: cellulitis, pharyngitis, scarlet
fever + complications: articular (acute rheumatic fever), cardiac (rheumatic carditis), renal (glomerulonephritis)
– Streptococcus pneumoniae: pneumonia, bronchopneumonia, meningitis
– Oral (viridans) streptococci: Streptococcus mutans, Streptococcus sanguis, Streptococcus anginosus (dental caries, periodontal disease + septicaemia, endocarditis)
”Strep throat” – Pharyngitis with Streptococcus pyogenes: left – petechiae; right – pus deposits
Periodontal disease, dental caries
Laboratory diagnosis of Streptococcal Infections: Gram stained smears
• Cocci: – Round / ovoid shape; arranged in chains / pairs– Gram positive – Aerobic growth (+anaerobic)
• + Streptococcus pneumoniae: pairs of encapsulated cocci
Streptococcus pyogenes: Gram stained smear:ovoid Gram positive cocci, arranged in chains
Streptococcus mutans – Gram stained smear
Streptococcus pneumoniae – Gram stained sputum smear
Laboratory diagnosis of Streptococcal Infections: Innoculation of culture media
Innoculation on blood agar:• Colonial morphology:
– Str.pyogenes: small, pinpont, 0.5 µM diameter, transparent– Str.pneumoniae, Str.viridans: small, smooth, flat/depressed
center (autolysis)
• Type of hemolysis: - β-hemolysis - complete digestion of red blood cell contents
surrounding colony e.g. Streptococcus pyogenes- α-hemolysis - partial lysis – incomplete hemoglobin digestion →
green or brown (conversion of hemoglobin to methemoglobin) e.g. Streptococcus viridans, Streptococcus pneumoniae
Streptococcus pyogenes on blood agar
Streptococcus pneumoniae on blood agar
Left: Alpha hemolysis – Streptococcus pneumoniaeRight: Beta hemolysis – Streptococcus pyogenes
Staphylococcus / Streptococcus (?)