Bacteriological diagnosis of infections caused by bacteria of

Staphylococcus and Streptococcus genera - Part One -

http://www.slideshare.net/DanaSinzianaBreharCi/staphylococcus-streptococcus-bacteriological-diagnosisi

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 i.e. purple (violet)– 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 - clinical significance -

• Community & Hospital acquired infections:

– Skin & subcutaneous tissues: foliculitis, abscesses, furuncles, carbuncles

– otitis, synusitis, pneumonia– Osteomyelitis, septic arthritis– Gentio-Urinary: cystitis, prostatitis, pielonephritis, renal

abscesses– Cardiovascular: endocarditis, phlebitis, sepsis– Digestive: Food poisoning – Nervous system: meningitis, encephalitis

Genus Staphylococcus: Steps of bacteriological diagnosis

• Collection of specimens (e.g. pus, pharyngeal exudate, urine, stool, etc)

• Macroscopic examination• Microscopic examination• Inoculation of culture media• Pathogenicity tests• Biochemical tests• Agglutination tests • Antimicrobial susceptibility tests (antibiogram)

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

Pharyngeal, naso-pharingeal exudatePatient:

– 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 uvulawithout 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

Genus Staphylococcus: Steps of bacteriological diagnosis

• Collection of specimens (e.g. pus, pharyngeal exudate, urine, stool, etc)

• Macroscopic examination• Microscopic examination• Inoculation of culture media• Pathogenicity tests• Biochemical tests• Agglutination tests• Antimicrobial susceptibility tests (antibiogram)

Laboratory diagnosis of Staphylococcal Infections: Gram stained smear from specimen (e.g. pus)

• White blood cells + Gram positive cocci • Shape: spherical• aglomerated in clusters / + pairs / + isolated• Location: both intra- and extracellular• Size: 0.5-1 µM

Staphylococcus: Gram staining biological product (sputum)

Staphylococcus: Gram staining

Genus Staphylococcus: Steps of bacteriological diagnosis

• Collection of specimens (e.g. pus, pharyngeal exudate, urine, stool, etc)

• Macroscopic examination• Microscopic examination• Inoculation of culture media• Pathogenicity tests• Biochemical tests• Agglutination tests• Antimicrobial susceptibility tests (antibiogram)

Laboratory diagnosis of Staphylococcal Infections: Inoculation 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, non-hemolytic

”Golden” colonies: Staphylococcus aureus

S.aureus golden, hemolytic colonies on blood agar

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 →S.aureus: Fermentation of mannitol

→colour of medium changes from pink to yellow (further identification step) – difference between S.aureus and other staphylococci

Mannitol Salt Agar (Chapman)

- high salt concentration supports growth of Staphylococcus / inhibits Streptococcus

- S.aureus: mannitol fermentation – changes the colour of the medium from pink to yellow

Chapman agar – mannitol fermentation (yellow) and no fermentation (pink)

Genus Staphylococcus: Steps of bacteriological diagnosis

• Collection of specimens (e.g. pus, pharyngeal exudate, urine, stool, etc)

• Macroscopic examination• Microscopic examination• Inoculation of culture media• Pathogenicity & Biochemical tests• Agglutination tests• Antimicrobial susceptibility tests (antibiogram)

Laboratory diagnosis of Staphylococcal Infections: Pathogenicity & biochemical tests

• Hemolysins – already discussed • Mannitol fermentation – already discussed• Catalase• Coagulase• Fibrinolysin• Biochemical tests • Bacteriophage typing

The Catalase test

• Principle: the enzyme catalase decomposes hydrogen peroxide (H2O2) into water and oxygen:

2H2O2 →2H2O + O2 (gas bubbles)

• 2-3 drops of hydrogen peroxide placed directly on a colony

• POSITIVE TEST: rapid effervescence

• differentiates Staphylococcus (+) / Streptococcus (-)

The Slide Coagulase test

• Principle: the coagulase of Staphylococcus aureus (aka ”clumping factor”) converts fibrinogen into fibrin →clot

• Procedure: – 2 drops of saline solution in 2 circles drawn on glass slide – Emulsify colony in each of the 2 circles – 1 drop of plasma (rabbit plasma with EDTA) in one circle– 1 drop of water in the other circle (control)– Rock slide back and forth & observe agglutination

• POSITIVE TEST: white precipitate & agglutination in 10-15 sec (control should remain smooth)

Slide and tube coagulase test

The fibrinolysin test

• Principle: fibrinolytic enzymes (e.g. the staphylokinase of S.aureus) can dissolve fibrin clots

• Procedure: • 3 tubes: CaCl2 + plasma → fibrin clot• Tube 1: add nothing else (clotting control)• Tube 2: inoculum of S. epidermidis strain; homogenize

with the loop, incubate at 37o C, 1-4 hours – fibrin clot not dissolved = NEGATIVE test

• Tube 3: inoculum of (suspected) S.aureus strain; homogenize inoculum with loop, incubate; clot slowly lysed = POSITIVE test

Testing for Enzyme Systems

• Final characterization of unknown bacterial isolate by testing for characteristic enzyme systems

• Method: re-inoculation of isolated colony (primary culture) into a series of culture media containing specific substrates and chemical indicators

• Principle: detection of – pH changes produced by utilization of substrates / – colour changes produced by specific by-products

• Challenge: Selection of appropriate sets of characteristics to allow bacterial group identification

Biochemical tests (testing for enzyme systems)

• The API Staph System (bioMerieux) – identification of 23 species of staphylococci

• 19 microampules containing dehydrated substrates and/or nutrient media

Procedure:- make a saline suspension of the organism from isolated colony- place staph strip in a tray with small amount of water to provide

humidity during incubation- dispense 2-3 drops of bacterial suspension in each microampule

with sterile pipette- cover tray and incubate aerobically for 18-24 hours at 35-37

degrees Celsius- seven-digit profile number obtained and used to determine the

identity of the organism (match to profile numbers from database)

Staphylococcus spp – biochemical tests

Genus Staphylococcus: Steps of bacteriological diagnosis

• Collection of specimens (e.g. pus, pharyngeal exudate, urine, stool, etc)

• Macroscopic examination• Microscopic examination• Inoculation of culture media• Pathogenicity & Biochemical tests• Agglutination tests• Antimicrobial susceptibility tests (antibiogram)

Rapid agglutination tests – detection of clumping factor of S.aureus

(Bacterio)phage typing – identification of strains causing epidemic clusters

• (Bacterio)phage = virus that infects bacterium – specificity allows id. of bacterial strains

• Bacterial culture+Grid drawn on lid of Petri dish

• 1 drop of different phage cultures in each quadrant & incubation

• Bacterial culture dissolved by respective bacteriophage – epidemiologic utility (identify source of epidemics)

Antimicrobial susceptibility• 1950-1960: emergence of strains resistant to antibiotics• MRSA (Methicillin Resistant S.aureus)• Mechanisms:

– enzyme which destroys β-lactam antibiotics (β-lactamase)– decrease of bacterial wall permeability

• Methicillin resistance = resistance to ALL β-lactam antibiotics (penicillins, cephalosporins, monobactams and carbapenems)

Gram positive cocci

• Family: Micrococaceae

• Genera:– Staphylococcus– Micrococcus– Stomatococcus– Planococcus

• Family: Streptococacceae

• Genera:– Streptococcus– Enterococcus– Aerococcus– Gemella– Leuconostoc– Pediococcus– Lactococcus

Classification of streptococci

Criteria:

• I. Type of hemolysis produced by bacterial growth on blood agar

• II. Antigenic structure (Lancefield)

Classification of streptococci according to type of hemolysis

• β-hemolytic streptococci:– Complete, clear hemolysis (medium around the colony is

transparent = bacterial growth produced complete digestion of red blood cells in the blood agar) e.g. Streptococcus pyogenes

• α-hemolytic streptococci:– partial hemolysis (medium around the colony is translucent and

greenish = bacterial growth produced incomplete digestion of hemoglobin in the blood agar (conversion of hemoglobin to methemoglobin) e.g. Streptococcus viridans, Streptococcus pneumoniae)

• Non-hemolytic streptococci

Blood agar: Enterococcus fecalis (non-hemolytic/variable) and

Streptococcus pyogenes (hemolytic)

Classification of streptococci according to antigenic structure (Lancefield grouping)

Rebecca Lancefield (1895-1981)(American microbiologist at the Rockefeller Institute for Medical

Research)

• based upon the C polysacharidic antigen (group-specific) in bacterial wall → groups A – H and K-V

• based upon M and T proteins (type specific) → over 80 types of group A streptococci

• !! Lancefield grouping does not include streptococci lacking group antigens e.g. Str.pneumoniae, Str.viridans, etc.)

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)

Streptococcus pyogenes- clinical significance -

• Acute, respiratory infections: pharyngitis, scarlet fever + complications: articular (acute rheumatic fever), cardiac (rheumatic carditis), renal (glomerulonephritis)

• Skin infections: erysipelas, impetigo, intertrigo, pemfigus, celulitis, abscesses + complications: sepsis

Streptococcus pyogenes: Steps of bacteriological diagnosis

• Collection of specimens (e.g. pus, pharyngeal exudate, content of vesicles, CSF, urine, etc)

• Microscopic examination• Inoculation of culture media• Biochemical tests• Agglutination tests • Antimicrobial susceptibility tests (antibiogram)

”Strep throat” – Pharyngitis with Streptococcus pyogenes: left – petechiae; right – pus deposits

Erysipelas – streptococcal infection of the dermis and superficial lymph vessels

Impetigo – non-bulous and bulous

Streptococcus pyogenes – Microscopic examination -

Gram stained smears:• Cocci:

– Round / ovoid shape; arranged in chains / pairs

– Gram positive – Aerobic growth (+anaerobic)

Streptococcus pyogenes: Gram stained smear:ovoid Gram positive cocci, arranged in chains

Streptococcus – Gram stained smear

Streptococcus pyogenes- Cultivation & isolation -

• Blood containing media e.g. blood agar, Todd-Hewit broth, SSP (selective medium for streptococci and pneumococci)

• Most frequently: – (Initial inoculation of selective medium (Pick) – favours growth

and multiplication of streptococci and inhibits other bacterial species)

– ↓– Reinoculation on 5% sheep blood agar

Streptococcus pyogenes- identification -

• Colonial characters: – small, pinpont, 0.5 µM diameter, transparent– β-hemolysis - complete digestion of red blood cell contents

surrounding colony

• Group identification: – bacitracin sensitivity test – group A streptococci are bacitracin

sensitive / other streptococci are resistant

Bacitracin sensitivity test

• used to determine the effect of a small amount of bacitracin (0.04 U) on an organism. 

• Streptococcus pyogenes (group A)  is inhibited (minimum 10 mm inhibition diameter) by the small amount of bacitracin in the disk; other beta-hemolytic streptococci usually are not

Blood agar platesLeft: Staphylococcus; Right: Streptococcus

Staph. aureus - mannitol fermentation (left side, left plate) Staph.epidermidis - no mannitol fermentation (right side, left plate)

Streptococcus – plate on the right

To be continued....