Gram Positive Cocci - Lecture 1-4 Flashcards
1
Q
Micrococcus - Overview
A
- closely resembles staphylococci (often confused)
- Skin coloniser - found on skin, face, arms, hands, legs, oropharynx.
- non-motile, obligate aerobe - requires oxygen
- catalase positive, oxidase positive - key for identification
2
Q
Micrococcus - Morphology & Identification
A
Gram Stain: Gram positive cocci (purple)
- occurs in pairs or terads
- large cocci (1-2um)
Blood Agar Growth & Colony Morphology:
- Pigmented colonies - yellow, orange or pink
- non-haemolyic - no blood cell breakdown
- large, convex (domed) colonies.
3
Q
Micrococcus - Clinical Significance
A
- Ubiquitous in the environment - found everywhere
- temporary resident ofhuman skin and mucous membranes
may appear in human and veterinary samples:- common contaminant in lab cultures
- potential pathogen in immunocompromised individuals
4
Q
Micrococcus - Pathogenic Potential (Rare)
A
- usually non-pathogenic, acts as a saprophyte
risk in immunocompromised patients - can cause:- pnuemonia
- septic shock
5
Q
Staphylococcus - Description
A
- found in human nasal cavity, other mucous membranes, skin
- facultative anaerobes - will grow in air and co2
- around 47 species and 23 sub-species present
38 of these are CoNS
coagulase production distinguishes the virulent S. auerus from less virulent CoNS - gram stain shows gram positive cocci in clusters
6
Q
Staphylococcus - Clinical Significance
A
- may be isolated from most clinical speciments (normal flora, coloniser, or pathogen)
- pathogens usually coagulase positive species (S.auerus, s.intermedius)
- CoNS (S.epidermidis, S.haemolyticus, S.saprophyticus) are increasingly linked to hospital infections or medical pocedures
enterotoxigenic strains can cause food poisioning
7
Q
Staphylococcus Aureus - Description
A
- carried in the anterior nares of around 30% of healthy adults and on the skin of around 20%.
- carriage rates are higher in hospital patients and staff
- S.auerus infections are more prevalent in carriers, usually caused by the colonising strain.
8
Q
Staphylococcus Auerus - Pathogen of Humans
A
- can cuase infections from normal carriage sites (skin, nares) to other locations in the host and others.
- causes a wide range of skin infections, healthcare-associated infection (HAIs).
- MRSA and VRSA are linked to surgical wound infections, UTIs, pneumonia.
- some strains produce toxins leading to gastroenteritis, scalded skin syndrome and toxic shock syndrome.
9
Q
Staphylococcus Aureus - Infections
A
- Skin infections:
- folliculitis
- boils
- cellulitis
- impetigo
- post-operative wound infections
- toxic shock syndrome
- bacteraemia
- osteomyelitis
- endocarditis
10
Q
Staphylococcus Epidermidis - Description
A
- 2nd most commonly isolated Staph species
- 50-80% of all CoNS isolates
- normal inhabitant of human skin and mucosal membranes
- has a capsule
capable of producing biofilms
11
Q
Staphylococcus Epidermidis - clinical signficance
A
- may cause infections in IVDU
- potential pathogen in hospital environments due to the wide use of medical implants and devices
- infection of indwelling protheses & intravascular devices:
- heart valves
- pacemakers
- prosthetic joints
- shunts
12
Q
Staphylococcus heamolyticus - Description
A
- normal flora of human skin )axillae, perineum, inguinal areas)
- coagulase negative staphylococcus (CoNS)
- lacks major virulence factors of other staphylococci
- known for acquiring multi-drug resistance
13
Q
staphylococcus haemolyticus - clinical significance
A
- 2nd most common CoNS species in human infections
- affects immune-compromised patients and those with implanted medical devices
- causes:
- septicemia
- endocarditis
- wound, bone and joint infections
- UTIs
14
Q
Staphylococcus saprophyticus – Description
A
- normal flora of the perineum, rectum, urethra, cervix and GIT
- found in 40% of young, sexually active women as part of geniurinary flora
- coagulase-negative staphylococcus (CoNS)
15
Q
Staphylococcus saprophyticus – Clinical Significance
A
- 2nd most common cause of community-acquired UTIs (after E.coli)
- causes umcomplicated UTIs (dysuria, frequency), especially in young sexually active women
- must be identified to species level if isolated from urine
- can lead to complications like acute pyelonephritis, urethritis, epididymitis and prostatis.
16
Q
Staphylococcus lugdunensis – Description
A
- normal skin flora in humans
- coagulase negative staphylococcus
- accounts for around 3% CoNS isolated in clincal labs
- most isolates remain susceptible to many antimicrobial agents.
17
Q
Coagulase Test Overview
A
Principle: coagulase has a prothrmbin-like acitvity that converts fibrinogen to fibrin, causing blood plasma to clot.
Types of Tests:
1. StaphTEX (Commercial Test): Latex beads with bound antibody added to a colony -> agglutination reaction if positive.
2. Tube test: mix rabbit plasma and nutrient broth with the colony -> if positive, contents solidify over several hours.
Note*
- commercial tests must include a negative control reagent to prevent false positives from Staph saprophyticus autoagglutination.
18
Q
Coagulase-negative species and pathogenic role
A
- causes opportunistic infections
leads to nosocomial infections (HAI - healthcare-associated infections)
incident has increased in the past 20 years, linked to: - higher use of prosthetic devices
increased number of immunocompromised (IC) patients in hospital.
19
Q
Novobiocin Sensitivity - Purpose
A
- helps differentiate between S.epidermidis and S.saprophyticus
- used to distinguish coagualse negative staph (CoNS)
20
Q
Novobiocin Sensitivity - Results
A
S.saprophyticus - Resistant
S.epidermidis and other CoNS - Sensitive
21
Q
Staphylococci on MSA - Purpose
A
- selective: high salt concentration allows growth of staphs
differential - mannitol fermentation causes a colour change in the pH indicator phenol red.
22
Q
MSA Results for staphs
A
- s.auereus, s.saprophyiticus, s.haemolyticus -> mannitol fermentation -> acid production -> phenol red turns yellow
- S.epidermidis -> no mannitol fermentation -> no acid production -> phenol red remains red.
23
Q
Streptococcus - Description
A
- gram positive cocci in pairs or chains, catalase-ngative
facultative anaerobes, require CO2 and enriched media (blood or choc agar)
classified by heamolysis patterns and lancefield typing
24
Q
differentiating staphs from streps
A
gram stain morphology:
- staphs -> clusters
- streps -> chains
s.pnuemoniae -> diplococci (pairs)
colony size:
- streps have much smaller colonies than staphs.
25
catalase test - distinguishing staphs from strep
- add 3% hydrogen peroxide to a glass slide
- catalase positive - bubbles (staphs)
- catalse negative - no bubbles (streps)
26
Lancefield Typing - Principle and Method
- B-heamolytic streps posses carbohydrate group specific antigens on their cell wall (Lancefield Groups)
- slide agglutination test used for grouping:
- can be done on primary plate with few colonies
- enzymes expose antigens
- sample reacts with group-specific antibody-coated latex beads
27
Groupable Strep (B-heamolytic) - common pathogens
- Group A (S.pyogenes) - causes pharyngitis, septicaemia, impetigo, rheumatic fever, glomerulonephritis
- Group B (S.agalactiae) - causes peurperal sepsis, neonatal meningitis
Haemolysis as a guide:
- Groupable Streps - b- or y- heamolysis
- non-groupable streps - a-heamolysis
28
S.pyogenes (Group A Strep) - Clinical Significance
- affects all ages, peak incidence: 5-15yrs
- commonly found in human pharyngeal aspirates
- produces enzymes and toxins, leading to invasive disease
Types of infection:
- Non-invasive:
- pharyngitis
- skin infections (impetigo)
- Invasive:
- toxic shock syndrome (superantigen)
- necrotising fasciitis ("flesh eating bacteria")
- pyrogenic toxin production
29
S.pyogenes Identification
Colony morphology:
- small, translucent grey colonies
- large zone of B-heamolysis
Biochemical Tests:
- bactitracin-sensitive
- PYR positive
Lancefield Grouping:
- Group A typing antigen
30
PYR Test - differentiation
- PYR test alone does not differentiate S.pyogenes from enterococcus
- combine with aesculin hydrolysis
- s.pyogenes -> PYR positive, aesculin negative
- enterococcus spp. -> PYR positive, aesculin positive
31
S.agalactiae - Clinical Significance
- commonly found in the female genital tract (normal vaginal flora)
- causes neonatal meningitis and septicaemia
- high risk for newborns
32
S.agalactiae - Identification
Colony Morphology:
- larger, translucent, flat, glossy, grey-white colonies
- small zone of B-heamolysis
Biochemical tests:
- bacitracin-resistant
- CAMP test positive
33
CAMP Reaction – Principle & Method
Principle: Group B Streptococcus (GBS) secretes a protein that enhances the action of Staphylococcus aureus β-lysin, leading to increased RBC lysis.
Method:
Streak β-haemolytic strain of S. aureus down the center of a sheep blood agar plate.
Streak the test organism perpendicular to the S. aureus streak, stopping ~2mm away.
Multiple organisms can be tested on the same plate.
34
CAMP Reaction – Results
Positive result:
Arrowhead-shaped zone of enhanced haemolysis at the junction of the two organisms (GBS).
Negative result:
No enhancement of haemolysis (seen with other Streptococci).
Note: If plates are incubated in CO₂, S. pyogenes may also produce an arrowhead-shaped zone.
35
Enterococcus – Clinical Significance
- Genus established in 1984, distantly related to other Streptococci.
- Normal flora of the GIT and female genital tracts.
Disease states:
- Urinary tract infections
- Fecal contamination
- Opportunistic infections, especially endocarditis.
- E. faecalis is the most common clinical isolate (85%), with E. faecium being less common.
- Resistant to many Gram-positive antibiotics.
36
Enterococcus – Identification
Colony Morphology:
- 0.5-1 mm grey-white colonies.
- Usually non-haemolytic (γ), sometimes α-haemolytic.
Cellular Morphology:
- Gram-positive cocci in short chains (Gram stain).
- Optochin-resistant.
- Bile insoluble.
- Often confused with viridans streptococci.
- Can grow at temperatures between 10°C and 45°C.
- Growth on bile aesculin agar (BAT agar): Forms black precipitate.
37
Bile Aesculin Agar – Principle
- Bile salts inhibit most Gram-positive bacteria (selective agent) → tests for bile salt tolerance.
- Enterococci hydrolyze aesculin to aesculetin.
- Several organisms can hydrolyze aesculin, but only a few can do so in the presence of bile salts.
38
Bile Aesculin Agar – Chemical Reaction & Results
- Aesculetin combines with ferric citrate (differential agent) to form a black complex in the medium.
- Tests for aesculin hydrolysis.
39
Viridans Streptococci – Clinical Significance
Diverse group of species, S. mutans is the “prototype”.
Normal flora of the oral cavity and respiratory tract.
Infective endocarditis:
- Infection of heart valves.
- Occurs after tooth extraction, as oral bacteria enter the bloodstream.
- If heart valves are damaged, clots (vegetations) form, protecting bacteria from the immune system.
40
S. pneumoniae – Clinical Significance
- Carried asymptomatically in the nose and throat of humans.
- Most common cause of community-acquired pneumonia.
- Also causes otitis media, bacteraemia, and meningitis.
- Capsule is crucial for virulence (over 90 capsular types).
Other terms:
- Diplococcus (Pneumococcus).
- Presence in sputum = pneumococcal pneumonia.
- Presence in spinal fluid = pneumococcal meningitis.
41
S. pneumoniae – Identification
Colony Morphology:
0.5 mm, glistening, grey colonies.
α-haemolytic.
Young cultures: raised; center flattens with age to form "draughtsman colonies".
Mucoid appearance if capsule is present.
Cellular Morphology:
Gram-positive diplococci.
42
Differentiating S. pneumoniae and S. viridans – Bile Solubility Test
Bile Solubility:
- S. pneumoniae: Lyses in the presence of bile salts (positive, clear).
- S. viridans: Bile-insoluble (negative, cloudy/turbid).
- Control: Tube with saline (no bile salts) is used for comparison.
Time: Rapid results (15 minutes).
43
Differentiating S. pneumoniae and S. viridans – Optochin Sensitivity
- S. pneumoniae: Sensitive to optochin after overnight incubation.
- S. viridans: Resistant to optochin.
Growth on BA:
- S. pneumoniae shows α-haemolysis and optimum sensitivity to optochin.
