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 (?)